Patent Application
20220184132
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is SEQUENCE_LISTING_790161_402USPC. The text file is 115 KB, was created on Aug. 4, 2021, and is being submitted electronically via EFS-Web.
The present disclosure relates to cell culture media and cell culture media supplements, which are non-human-animal-product free. More particularly, the present disclosure relates to platelet lysate obtained from a mixture of umbilical cord blood (UCB) and maternal blood (MB) platelet-rich plasma. Further, the present disclosure relates to a method of preparing said platelet lysate.
Cell and tissue culture media are typically composed of mineral salts, amino acids and vitamins to aid cultivation and propagation of cells under ex-vivo conditions. Generally, these media are supplemented with non-human, animal-derived additives such as non-human animal-derived Fetal Bovine Serum (FBS). An alternative means of supplementing cell culture media is by means of feeder layer cells.
However, the use of FBS is marred with challenges that include: (a) a cocktail of undefined qualitative and quantitative composition; (b) a considerable ethical concern; (c) global supply and availability of FBS can be highly fluctuating; and (d) batch-to-batch variation of FBS. Further, use of FBS in therapeutic applications is a major and undesirable risk for patients in clinical settings and discouraged by regulatory authorities to limit the risk of zoonoses and xenogeneic immune reactions in the transplanted host.
Thus, there is a need to find an appropriate replacement, with the required efficacy and efficiency for culturing cells such as stem cells and cells for therapeutic applications. One such growth supplement may be platelet-derived products, e.g., platelet lysates.
Platelet-based biomaterials such as platelet gel, platelet glue and platelet rich plasma have been reported to be used in the treatment of chronic ulcers and are a potential source in orthopaedics to facilitate healing and enhancing bone-grafting following implantation (refer Shaheen A et al., Austin J Dermatolog, 2018, 5(1):1085-1094, Wellington K Hsu et al., J Am Acad Orthop Surg 2013; 21:739-748, and Mlynarek R A et al.Am J Orthop. 2016; 45(4):290-294, 326).
The methods for isolation and enrichment of platelet fraction from blood and further preparation of platelet lysates have relied upon centrifugation as a means to segregate platelet-rich plasma fraction from a blood sample, such as peripheral blood from a human subject from the cell pellet and platelet-poor plasma fraction, for example.
CN106236779 discloses a method for preparing platelet-rich plasma (PRP) from cord blood.
Bernardi et al. (Journal of translational medicine, 2017; vol. 15,1 90. 1, doi:10.1186/s12967-017-1185-9), discloses that the production method used to release platelet factors significantly affects the enrichment in growth factors and overall product performance.
EP2757879A1 discloses a method for producing a platelet lysate with a maximum level of safety by subjecting platelets to a pathogen inactivation.
US9700583B2 discloses a method of autologous mesenchymal stem cells (MSCs) isolation from a patient, expansion in presence of platelet rich paste obtained by centrifugation and subsequent implantation into said patient.
Shirzad et al. (Cell journal, 2017; 19(3):403-414), discloses culture supplements devoid of animal-derived products and provides umbilical cord blood-platelet lysate (UCB-PL) as a standard substitute for FBS and human peripheral blood-PL (PB-PL).
Kwok et al. (Br J Haematol. 2007; 137(5):468-74), discloses that the presence of maternal plasma/human serum albumin (HSA) in the efficient purification and culture of CD34+ cells derived from human umbilical cord blood.
US20110123503A1 discloses platelet fractions which can be obtained from placental blood.
The above described methods for preparation of platelet fractions, including platelet lysates have drawbacks and problems in terms of loss of logistics, costs and amount of cellular (platelet) damage due to the use of centrifugation/apheresis manufacturing procedures on blood products. Further, the loss of discarded umbilical cord blood (UCB) or discarded maternal blood (MB) isolated for testing or otherwise, which potentially comprises a plethora of growth factors and differentiation factors that may be exploited for therapeutic applications in cell and tissue cultures meant for implantation into patients and beyond has potential economic benefits.
Gifford et al. (PLoS ONE, 2018; 13(1): e0190827), discloses the logistical complications and potential cellular damage associated with centrifugation/apheresis manufacturing of blood products.
Hence, there remains a problem of finding an appropriate method for platelet fraction isolation, providing a viable and economical source for isolation.
All publications mentioned in this document are incorporated fully by reference.
In an aspect of the present invention, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least one sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood
(MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets.
In another aspect of the present invention, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the UCB+MB platelet-rich plasma mixture at a temperature in a range of −70° C. to −86° C. for a time in a range of 24 hours to 96 hours to obtain a frozen UCB+MB platelet-rich plasma mixture; and (h) subjecting the frozen UCB+MB platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets.
These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.
For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.
The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.
Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.
The term “including” as used herein means “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.
The term “at least” as used herein means not less than the following amount.
The term “lysate” as used herein includes cellular lysates, platelet lysates, plasma and combinations thereof that are procured following cellular lysis. Cellular lysis may be brought on by freeze/thaw cycles, osmotic changes, other physical and chemical means known in the field.
The term “platelet-rich plasma” (PRP) as used herein means the less light plasma portion of the blood that comprises platelets in a concentration above one million per microliter.
The term “platelet lysate” as used herein means cell lysates produced from regular platelet transfusion units by lysis.
The term “platelet” as used herein refers to cells which are small a-nucleated structures of hematopoietic origin which contribute to homeostasis and wound healing by secreting growth factors and cytokines. They are produced by the fragmentation of megakaryocytes and released into the bloodstream, where they circulate for 7-10 days before being replaced.
The term “subject” as used herein refers to any vertebrate animal and does not merely cover human. Human subjects have been used to exemplify the invention but, said exemplification should not be considered in any way limiting to the scope of the subject matter as covered under the term subject.
The term “umbilical cord blood” (UCB) as used herein means the blood that remains in the placenta and in the attached umbilical cord after childbirth. Cord blood is collected because it contains stem cells, which can be used to treat hematopoietic and genetic disorders. Generally, a lot of this rich biological resource is discarded. The preferred source is human. The term “in-vitro assays” depict the assays which can be done in in-vitro. The application of such assays can range from therapeutics, diagnostics to predictive studies for development.
The process for umbilical cord blood (UCB) collection entails: (a) confirming the identity of a subject, (b) cleaning the segment of the umbilical cord with 10% povidone Iodine and 70% alcohol (Ethyl Alcohol/Isopropyl Alcohol) thrice alternately, swabbing away from the collection area, before collection, (c) after spirit evaporates, removing outer gloves to prevent contamination, (d) holding cord blood collection bag with sterile inner gloves, (e) inserting one end of a needle in the umbilical cord vein near the cord clamp and the other end into a blood collection bag, (f) gently and properly mixing the cord blood flowing into the collection bag with an anticoagulant, (g) once umbilical cord appears empty and whitish and all blood has been removed, stopping the blood link through the needle, checking for leakage, if any, and cleaning the blood collection bag with the collected umbilical cord blood (UCB) with sterile gauze. The preferred source is human.
The term “maternal blood” (MB) as used herein means the blood collected from a mother pre- and post-delivery. The maternal blood (MB) collection may take place at a time immediately before/after cord blood collection, at the time of admission for delivery (after initiation of labour) or before transfusion/infusion of any intravenous fluid (colloids/crystalloids/blood products). The preferred source is human.
The term “umbilical cord blood and maternal blood” (UCB+MB) as used herein means any combination of an umbilical cord blood and maternal blood. Said combination may come from autologous and/or allogenic sources. The preferred source is human.
The term “sedimentation” as used herein refers to the tendency for heavier particles in suspension to settle out of the fluid in which they are entrained and come to rest against a barrier like bottom of a container. Such a motion through the fluid may be in response to the forces acting on them, which may include, gravity, centrifugal acceleration, or electromagnetism.
The term “at least one sedimentation” as used herein refers to a minimum of one sedimentation.
The term “at least two sedimentations” as used herein refers to a minimum of two sedimentations.
The term “sedimentation mixture” as used herein means any mix of substances that leads to and/or aids a sedimentation process, where heavier components of the mixture settle on the bottom, due to gravity, leaving the lighter components amenable for separation such as by decantation or pipetting.
The term “sedimentation reagent” as used herein means any substance that aids coagulation or aggregation of particles or components in suspension to form heavier particles that then come to rest against a barrier like bottom of a container due to the forces acting on them such as gravity. As used herein, a sedimentation reagent such as Ficoll-Hipaque, Hespan®, Pentastarch, and combinations thereof.
The term “frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture” as used herein means the mixture obtained by freezing said mixture at a temperature in a range of −70° C. to −86° C. for a time in a range of 24 hours to 96 hours.
The term “freeze-thaw cycle” as used herein refers to a process of rapid freeze (bringing to low temperature) and slow thaw (bringing to high temperature) that results in cell death and/or cell lysis. Often repeated a set number of times according to the size and nature of cells being treated. As used herein the freeze-thaw cycles may range from 1 to 5, and the temperature for freezing ranges from −70° C. to −86° C. and the temperature for thawing ranges from 30° C. to 40° C.
The term “platelets/ml” as used herein refers to the number of platelets per millilitre of a fluid. Preferably said fluid is plasma.
The term “a combination of umbilical cord blood derived platelets and maternal blood derived platelets” as used herein refers to mixing or combination of platelets derived from processing umbilical cord blood and maternal blood, whether allogenous or autogenous by origin, and mixed in a ratio in a range of 10:1 to 30:1, and preferably, 10:1 to 26:1.
The term “infectious diseases” as used herein refers to diseases caused by pathogens such as microbes including HIV I and II, HBs, HCV, Syphilis, Mycoplasma etc.
The term “enriched” as used herein refers to enhancement in concentration of an amount of a component per volume containing said component following processing steps. For platelet-rich plasma as used herein, to obtain umbilical cord blood and maternal blood (UCB+MB) ‘enriched’ platelet lysate, the platelets in platelet-rich plasma obtained by combination of umbilical cord blood and maternal blood (UCB+MB) are in a range of 0.3×109to 1.5×109 platelets/ml.
The term “culturing” as used herein refers to maintaining (tissues, cells, bacteria, etc.) in conditions suitable for growth, propagation, expansion, differentiation and the like, in a specially prepared nutrient medium under supervised conditions.
The term “cells” as used herein refers to any type of stem cells, progenitor cells and differentiated cells. Preferably, the cells are selected from a group consisting of progenitor cells, osteoblasts, chondrocytes, buccal epithelial cells, dermal culture, cord tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and cardiomyocytes. The cells are preferably human by origin.
The term “therapeutic applications” means applications including, but not limited to, mesenchymal stem cells derived from human umbilical cord tissue, osteoblasts differentiated from bone marrow derived mesenchymal stem cells, cardiomyocytes differentiated from human umbilical cord tissue derived mesenchymal stem cells, islets cells of pancreas differentiated from human umbilical cord tissue derived mesenchymal stem cells, chondrocytes from human cartilage biopsy and buccal biopsy derived dermal fibroblasts. The therapeutic applications are further incorporated with but not limiting to, certain biomaterials or cell-gel techniques or methods thereof.
Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
To address the problems encountered with use of FBS, the present disclosure provides an optimized non-animal origin cell and tissue culture supplement in the form of an umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate. Further, the present disclosure provides a method for preparing a lysate, wherein the lysate is the umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.
The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.
In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least one sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB×MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets. In another embodiment of the present disclosure, the umbilical cord blood (UCB) sedimentation mixture is subjected to at least two sedimentations. In yet another embodiment of the present disclosure, the storing is done at a temperature in a range of −70° C. to −86° C. to obtain the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture. In a further embodiment of the present disclosure, the storing is done for a time in a range of 24 hours to 96 hours. In a supplementary embodiment of the present disclosure, the lysate is selected from a group consisting of cell lysate, platelet lysate, plasma and combinations thereof. In an alternate embodiment of the present disclosure, the lysate is a platelet lysate.
In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of −70° C. to −86° C. for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets. In another embodiment of the present disclosure, lysate is selected from a group consisting of cell lysate, platelet lysate, plasma and combinations thereof. In an alternate embodiment of the present disclosure, the lysate is a platelet lysate.
In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to at least one sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (d) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to at least one sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (e) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (c) and the maternal blood (MB) derived platelet-rich plasma of step (d) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (f) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to obtain a frozen umbilical cord blood and maternal blood (UCB−MB) platelet-rich plasma mixture; and (g) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the lysate is a platelet lysate, wherein the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent is in a volume ratio of 3:1 to 9:1, or in a volume ratio of 4:1 to 8:1, or a volume ratio of 4.5:1 to 6:1, or in a volume ratio of 5:1. In another embodiment of the present disclosure, the lysate is selected from a group consisting of cell lysate, platelet lysate, plasma and combinations thereof. In an alternate embodiment of the present disclosure, the lysate is a platelet lysate. In another embodiment of the present disclosure, the at least one sedimentation is carried out at a temperature in a range of −70° C. to −86° C. for a time in a range of 24 hours to 96 hours. In a supplementary embodiment of the present disclosure, the umbilical cord blood (UCB) sample and maternal blood (MB) sample are independently subjected to an initial platelet count by withdrawing aliquot of 100-200 μl.
In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood
(UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture;
(g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of −70° C. to −86° C. for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the first, the second and the third sedimentation are carried out for 25-55 minutes, or for 32-42 minutes, or for 35 minutes. In another embodiment of the present disclosure described herein, the storing of the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture is at a temperature of −86° C. for a time of 48 hours. In an auxiliary embodiment of the present disclosure described herein, the cord blood (UCB) sample and maternal blood (MB) sample are independently subjected to an initial platelet count by withdrawing aliquot of 100-200 μl. In an additional embodiment of the present disclosure as described herein, the lysate is selected from a group consisting of cell lysate, platelet lysate, plasma and combinations thereof.
In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of −70° C. to −86° C. for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the umbilical cord blood (UCB) sample is at least 100 ml and the maternal blood (MB) sample is in a range of 15ml to 20 ml, or in a range of 80 ml to 120ml and the maternal blood (MB) sample is in a range of 15 ml to 20 ml. In an auxiliary embodiment of the present disclosure, the storing of the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture is at a temperature of -86° C. for a time of 48 hours. In a supplementary embodiment of the present disclosure, the umbilical cord blood (UCB) sample and maternal blood (MB) sample are independently subjected to an initial platelet count by withdrawing aliquot of 100-200 μl. In an ancillary embodiment of the present disclosure, lysate is selected from a group consisting of cell lysate, platelet lysate, plasma and combinations thereof.
In an embodiment of the present disclosure, therein is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature of −86° C. for a time of 48 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the human umbilical cord blood (UCB) and maternal blood (MB) platelets are obtained from plasma which is considered as a bio-waste obtained after processing the cord blood. In an ancillary embodiment of the present disclosure, lysate is selected from a group consisting of cell lysate, platelet lysate, plasma and combinations thereof. Said bio-waste are discarded after delivery and tests. Further, said umbilical cord blood (UCB) and maternal blood (MB) are checked by infectious disease testing and only the samples free of contaminations and free from infectious diseases are selected for the preparation of platelet lysate.
In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of −70° C. to −86° C. for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture comprises platelets in a range of 0.3×109 to 1.5×109 platelets/ml, or 0.3×109 to 0.7 x109 platelets/ml, or 0.3×109 to 0.5×109 platelets/ml.
In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of −70° C. to −86° C. for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the mixing of umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) is done in a volume ratio having a range of 10:1 to 30:1, or 20:1 to 30:1.
In an embodiment of the present disclosure, there is provided a method for preparing a lysate as described herein, wherein the freeze-thaw cycles range from 1-5 or from 2-3.
In an embodiment of the present disclosure, there is provided a method for preparing a platelet lysate as described herein, wherein the freeze-thaw cycles range from 1-5 or from 2-3.
In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of −70° C. to −86° C. for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the at least one sedimentation reagent is selected from a group consisting of Ficoll-Hipaque, Hespan®, Pentastarch, and combinations thereof.
In an embodiment of the present disclosure, there is provided a method for preparing a lysate, said method comprising: (a) obtaining an umbilical cord blood (UCB) sample; (b) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (c) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (d) subjecting the plasma of step (c) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (e) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (f) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (d) and the maternal blood (MB) derived platelet-rich plasma of step (e) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (g) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of −70° C. to −86° C. for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (h) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, and wherein subjecting the umbilical cord blood (UCB) sedimentation mixture to a first, a second, and a third sedimentation is done for at least 30 minutes, or for a time period in a range of 30-45 minutes. In a complementary embodiment of the present disclosure, subjecting the maternal blood sample (MB) sample to sedimentation is done for a time in a range of 50-70 minutes.
In an embodiment of the present disclosure, there is provided a method for preparing a lysate as described herein, wherein the lysate obtained is a platelet lysate, and wherein the platelet lysate is filtered using a 0.22μ filter.
In an embodiment of the present disclosure, there is provided a method for preparing a lysate as described herein, wherein the lysate obtained is subjected to screening for presence of infectious diseases (ID). In an auxiliary embodiment of the present disclosure, the screening involves testing for microbial testing, mycoplasma testing, and infectious diseases (ID) viz., HIV I and II antibodies, HBs Ag (surface antigen), HCV antibodies and Syphilis antibodies.
In an embodiment of the present disclosure, there is provided an umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived platelet-rich plasma; and (b) a platelet lysate obtained from maternal blood (MB) derived platelet-rich plasma, wherein the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of 0.3×109 to 1.5×109 platelets/ml, for use in culturing cells. In an auxiliary embodiment of the present disclosure, the culturing cells are selected from a group consisting of progenitor cells, osteoblasts, chondrocytes, buccal epithelial cells, dermal culture, cord tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and cardiomyocytes.
In an embodiment of the present disclosure, there is provided an umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived platelet-rich plasma; and (b) a platelet lysate obtained from maternal blood (MB) derived platelet-rich plasma, wherein the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of 0.3×109 to 1.5×109platelets/ml, for use in therapeutic applications.
In an embodiment of the present disclosure, there is provided an umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived platelet-rich plasma; and (b) a platelet lysate obtained from maternal blood (MB) derived platelet-rich plasma, wherein the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of 0.3×109 to 1.5×109 platelets/ml, wherein said umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate is cryopreserved for a time in a range of 1 day to 12 months at a temperature in a range of −70° C. to −86° C. for use in culturing cells. In a further embodiment of the present disclosure, said umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate is cryopreserved for a time of 6 months at a temperature in a range of −86° C. for use in culturing cells, or for a time of 12 months at a temperature in a range of −86° C. for use in culturing cells. In an accompanying embodiment of the present disclosure, the umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate is for use in culturing cells, and wherein the cells are selected from a group consisting of progenitor cells, osteoblasts, chondrocytes, buccal epithelial cells, dermal culture, cord tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and cardiomyocytes.
In an embodiment of the present disclosure, there is provided an umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived platelet-rich plasma; and (b) a platelet lysate obtained from maternal blood (MB) derived platelet-rich plasma, wherein the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of 0.3×109 to 1.5×109 platelets/ml, wherein said umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate is cryopreserved for a time in a range of 1 day to 12 months at a temperature in a range of −70° C. to −86° C. for use in therapeutic applications.
In an embodiment of the present disclosure, there is provided an umbilical cord blood and maternal blood (UCB+MB) enriched platelet lysate comprising: (a) a platelet lysate obtained from umbilical cord blood (UCB) derived platelet-rich plasma; and (b) a platelet lysate obtained from maternal blood (MB) derived platelet-rich plasma, wherein the umbilical cord blood derived platelets and the maternal blood derived platelets have a combined platelet count in a range of 0.3×109 to 1.5×109 platelets/ml, wherein, the enriched platelet lysate is obtained by a method comprising: (i) obtaining an umbilical cord blood (UCB) sample; (ii) contacting the umbilical cord blood (UCB) sample with at least one sedimentation reagent to obtain an umbilical cord blood (UCB) sedimentation mixture; (iii) subjecting the umbilical cord blood (UCB) sedimentation mixture to a first and a second sedimentation to obtain plasma comprising umbilical cord blood (UCB) derived platelets; (iv) subjecting the plasma of step (iii) to a third sedimentation to obtain an umbilical cord blood (UCB) derived platelet-rich plasma; (v) obtaining a maternal blood (MB) sample, and subjecting the maternal blood (MB) sample to sedimentation to collect maternal blood (MB) derived platelet-rich plasma; (vi) mixing the umbilical cord blood (UCB) derived platelet-rich plasma of step (iv) and the maternal blood (MB) derived platelet-rich plasma of step (v) to obtain an umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; (vii) storing the umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture at a temperature in a range of −70° C. to −86° C. for a time in a range of 24 hours to 96 hours to obtain a frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture; and (viii) subjecting the frozen umbilical cord blood and maternal blood (UCB+MB) platelet-rich plasma mixture to freeze-thaw cycles to obtain a lysate from a combination of umbilical cord blood derived platelets and maternal blood derived platelets, wherein the umbilical cord blood (UCB) sedimentation mixture comprises the umbilical cord blood (UCB) sample and the at least one sedimentation reagent is in a volume ratio of 3:1 to 9:1, or 4:1 to 8:1, or 4.5:1 to 6:1, or 5:1.
Although the subject matter has been described in considerable detail with reference to certain examples and implementations thereof, other implementations are possible.
The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply.
The examples as presented herein describe the best working process of the present disclosure.
Umbilical cord blood (UCB) was collected from an identified subject. Before collection, the segment of the umbilical cord of said subject was cleaned with 10% povidone Iodine and 70% alcohol (Ethyl Alcohol/Isopropyl Alcohol) thrice alternately, swabbing away from the collection area. After the spirit evaporated, the outer gloves were removed to prevent contamination. Then the umbilical cord blood (UCB) collection bag was held with sterile inner gloves and a sterile needle was inserted into the umbilical cord vein near the cord clamp and said needle was connected to the umbilical cord blood (UCB) collection bag that held a sterile anticoagulant such as citrate-phosphate-dextrose solution. The flowing cord blood through the needle into the umbilical cord blood (UCB) collection bag was gently and properly mixed with the anticoagulant in the umbilical cord blood (UCB) collection bag. After the collection of maximal amounts of cord blood, till the umbilical cord ran empty and appeared whitish, multiple knots were tied at the opening end of the umbilical cord blood (UCB) collection bag after cutting the input from the needle with care and the collection bag was checked for leakage. The umbilical cord blood (UCB) collection bag was finally cleaned with sterile gauge pieces and used for transport to a cell processing centre. The collected umbilical cord blood (UCB) was transported to the cell processing centre at 18° C. to 28° C. within a period of less than 72hours post-collection. After receiving the sample, umbilical cord blood (UCB) bag was cleaned with 70% IPA and then it was sealed and labelled.
The accuracy and identity of the subject mother was confirmed. Maternal blood (MB) sample was collected in a sterile vacutainer tube with a sterile anticoagulant such as EDTA. The collection of maternal blood (MB) sample was carried out at the time of admission for delivery (after initiation of labour) or before transfusion/infusion of any intravenous fluid (colloids/crystalloids/blood products) and immediately before/after cord blood collection. The collected maternal blood (MB) was transported to the cell processing centre at 18° C. to 28° C. within a period of less than 72 hours post-collection. After receiving the sample, maternal blood (MB) vacutainer tube was cleaned with 70% IPA and then it was sealed and labelled.
Steps for preparation of platelet lysate from umbilical cord blood (UCB) and maternal blood (MB) samples is represented in a flow-chart (refer
For processing the umbilical cord blood (UCB) bag, a calculated volume of sedimentation reagent, such as Ficoll-Hipaque, Hespan®, Pentastarch, etc., was withdrawn under sterile conditions and injected into the umbilical cord blood (UCB) collection bag and kept on a rocking shaker for 5-10 minutes. A processing bag was labelled and attached to the said umbilical cord blood (UCB) collection bag. The said umbilical cord blood (UCB) collection bag was subjected to a first and then a second sedimentation for 35 minutes each so as to allow Rouleax formation of the red blood cells (RBCs) for better separation of the RBCs from the plasma. After said sedimentations, with the use of auto volume expresser, umbilical cord blood (UCB) platelet-rich plasma was collected in the umbilical cord blood (UCB) processing bag. Further, the umbilical cord blood (UCB) platelet-rich plasma in the umbilical cord blood (UCB) processing bag was subjected to a third sedimentation for another 35 minutes to separate out hematopoietic stem cells (HSCs) from the plasma with the use of auto volume expresser and the final umbilical cord blood (UCB) derived platelet-rich plasma was collected in a 50 ml conical tube.
At the same time as the processing of umbilical cord blood (UCB), the maternal blood (MB) sample was kept for sedimentation for 60minutes and then, the supernatant was collected to procure maternal blood (MB) derived platelet-rich plasma into a 50 ml conical tube.
The isolated umbilical cord blood (UCB) derived platelet-rich plasma and maternal blood (MB) derived platelet-rich plasma were mixed together and stored at −86° C. for 48 hours and further this mixture of platelet-rich plasma was taken through multiple cycles of freeze-thaw cycles ranging from 1-3 times to get to good quality platelet lysate (PL) called umbilical cord blood (UCB) plus maternal blood (MB) platelet lysate or UCB+MB platelet lysate. Said UCB+MB platelet lysate was filtered using a 0.22μ filter and tested for microbial sterility, mycoplasma and infectious diseases viz., HIV I and II antibodies, HBs Ag (surface antigen), HCV antibodies and Syphilis antibodies.
Using this method, from around 100 ml of umbilical cord blood (UCB), approximately 40 ml to 60 ml of umbilical cord blood (UCB) derived platelet lysate may be obtained and from around 15 ml to 20 ml of maternal blood, approximately 10 ml of maternal blood (MB) derived platelet lysate may be obtained.
Example 4 analyzed three different time points used for isolation of platelets from human umbilical cord blood (UCB) samples, namely, 20 minutes, 35 minutes and 60 minutes. Sample size for the experiment was 5 umbilical cord blood (UCB) samples. Samples 1 to 5 refer to the UCB obtained from five subjects.
Table 1 presents umbilical cord blood (UCB) samples initial and final platelet counts (platelets/ml) recovered with different time periods of sedimentation with a sedimentation reagent (Hespan® which is 6% hetastarch in 0.9% sodium chloride solution). A total of 5 sample data have been represented in triplicates in said table. Herein, quantification having average and standard deviation were calculated of platelet counts of UCB.
From the table above, one can deduce that at 35 minutes of sedimentation platelet recovery was more than 90%, while at 20 mins recovery was high but mixed population along with platelets and RBC, WBCs and at 60 mins platelet recovery was very less (less than 85%). Also, there was no impurity after the sedimentation process.
Example 5 analyzed volume ratio of cord blood along with sedimentation reagent used for isolation of platelets from human umbilical cord blood samples 2.5 ml UCB: 1 ml Sedimentation reagent(SDR)-Ratio-1; 5 ml UCB: 1 ml Sedimentation reagent (SDR)-Ratio-2; and 10 ml UCB: 1 ml Sedimentation reagent (SDR)-Ratio-3. Sample size for the experiment was 5 umbilical cord blood (UCB) samples. Each sample was divided into three parts.
Table 2 presents umbilical cord blood (UCB) samples initial and final platelet counts (platelets/ml) recovered with sedimentation using different volume ratios of UCB (ml) to sedimentation reagent (Hespan® which is 6% hetastarch in 0.9% sodium chloride solution). A total of 5 sample data have been represented in triplicates in said table. Herein, quantification having average and standard deviation were calculated of platelet counts of UCB.
*Inference: At different volume ratios Platelet recovery was carried out.
From the table above, one can deduce that at 5:1 ratio of UCB: sedimentation reagent, platelet recovery was more than 90%, while at 2.5:1 and 10:1 ratios, the recovery was less than 60%. Therefore, the ratio of UCB to sedimentation reagent plays a critical role in platelet recovery. The present Example highlights the experimental efforts to arrive at a specific ratio.
Table 3 below shows the recovery percentage of platelets through sedimentation process from maternal blood (MB)
It can be observed from the Table above that the recovery of samples from the sedimentation process was above 75% in case of maternal blood.
Blood Sample Collection:
Umbilical cord blood (UCB, about 150 ml) was drawn from the umbilical cord tissue of the five healthy subjects into the collection bag containing citrate-phosphate-dextrose as an anticoagulant. The umbilical cord blood (UCB) collection bag was then transported to the cell processing centre (CPC) at 18-28° C. along with 10 ml of maternal blood in (MB) vacutainer tube.
Isolation of Platelet Lysate from UCB and MB Using Sedimentation:
The method described in Example 3 was employed for isolation of platelets from UCB and MB to obtain UCB, MB and UCB+MB platelet lysates.
Isolation of Platelet Lysate from UCB Using Centrifugation:
The procedure followed has been taken from Shaheen A et al., Wellington K Hsu et al., and Mlynarek R A et al. (refer Shaheen A “Platelet Rich Plasma (PRP) for Treatment Non-Healing Ulcers: A Review Study,” Austin J Dermatolog, 2018, 5(1):1085-1094, Wellington K Hsu et al. “Platelet-rich Plasma in Orthopaedic Applications: Evidence-based Recommendations for Treatment,” J Am Acad Orthop Surg 2013;21:739-748, and Mlynarek R A et al. “Platelet-Rich Plasma (PRP) in Orthopedic Sports Medicine,” Am J Orthop. 2016 May;45(4):290-294, 326).
The aliquots from platelet-rich plasma from UCB and MB before and after sedimentation process showed that the average platelet count remained almost the same, even with the decrease in plasma volume.
Table 4 presents (a) the umbilical cord blood (UCB) derived platelet count before and after sedimentation; and (b) the maternal blood (MB) derived platelet count before and after sedimentation. A total of 5 sample data have been represented in triplicates in said table. Herein, quantification having average and standard deviation were calculated of platelet counts of UCB and MB respectively.
The average platelet counts and platelet recovery before and after sedimentation was calculated and presented in Table 4. From Table 4, one can infer that the average recovery of platelets from UCB and maternal blood by sedimentation method ranges from 94%-100%. It can be appreciated from the Table 4 that most platelets were recovered during the sedimentation process without major loss. Also, the average recovery of the platelets from UCB of the samples 1-5 is 99% and the average recovery of the platelets from MB is 97%.
The platelet counts obtained from UCB through the centrifugation process was measured for comparison with the sedimentation process.
Table 5 shows the UCB derived platelet counts and MB derived platelet counts before and after centrifugation. A total of 5 sample data have been represented in triplicates. Quantification having average and standard deviation were calculated of Platelet counts of UCB.
Average platelet counts and platelet recovery before and after centrifugation of umbilical cord blood (UCB) samples were calculated and have been shown in Table 5. From Table 5, one can infer that the average recovery of platelets from UCB by centrifugation method ranges from 47%-70%. Similarly, the recovery of MB derived platelets is also on a lower range. This states that most platelets were not recovered during the standard centrifugation process.
Table 6 provides a comparison of sedimentation versus centrifugation method of (UCB+MB) derived platelets and UCB derived platelets respectively.
It can be appreciated that the present process as disclosed herein provides significantly higher recovery than centrifugation method.
Further the collected platelets of UCB and MB through the process of sedimentation was further enriched by the process of centrifugation for 10 minutes to provide higher and concentrated yield of platelet which could have potential use for therapeutic purpose. The
Table 7 provides the data of the enrichment of the UCB and MB derived platelets.
As can be observed the average platelet count reached up to 1.46×109/ml.
HIV I/II ELISA was employed herein, which involved an immunosorbent enzyme assay which consists of recombinant protein for gp120, gp41 of HIV-I and gp36 of HIV-II bound to wells of microplate. During the course of assay, diluted controls and diluted specimens were added to the wells and incubated. HIV specific antibody (Ab), if present, binds to the antigens. After a thorough washing of the wells to remove unbound Ab and other serum components, standardized preparation of horse radish peroxidase—conjugate was added to each well. The conjugate preparation was then allowed to react with antibodies which bind to the assay wells on the basis of the specificity for antigenic determinants present within HIV antigens. After second thorough washing of the wells to remove unbound horseradish peroxidase-conjugated Ab, a substrate solution containing hydrogen peroxide and TMB was added to each well. A blue colour developed in proportion to the amount of HIV specific antibodies present, if any, in serum or plasma samples tested. This enzyme-substrate reaction was terminated by addition of sulfuric acid. The colour changes to yellow that have occurred in each well were then measured spectrophotometrically at a wavelength of 450 nm/630 nm. The sample was considered negative when OD value of the sample is less than the value of positive control.
An indirect antibody EIA assay for HCV ELISA was performed for detection of antibodies to HCV in human serum and plasma. It employed an immunosorbent enzyme assay which consists of recombinant protein for core and Ns3 protein and synthetic peptides corresponding to highly antigenic segments, Ns4 and Ns5 regions of the hepatitis C virus bound to wells of a microplate. During the course of the assay, diluted controls and diluted specimens were added to the wells and incubated. HCV specific Ab, if present, binds to the antigens. After a thorough washing of the wells to remove unbound Ab and other serum components, standardized preparation of horse radish peroxidase-conjugate was added to each well. The conjugate preparation was then allowed to react with antibodies which bind to the assay wells on the basis of the specificity for antigenic determinants present within HCV antigens. After second thorough washing of the wells to remove unbound horseradish peroxidase-conjugated Ab, a substrate solution containing hydrogen peroxide and TMB was added to each well. A blue colour develops in proportion to the amount of HCV specific antibodies present, if any, in serum or plasma samples tested. This enzyme-substrate reaction was terminated by addition of sulfuric acid. The colour changes to yellow was measured in each well spectrophotometrically at a wavelength of 450 nm/630 nm. The sample was considered negative when OD value of the sample was less than the value of positive control.
A solid phase ELISA for HBsAg detection based on sandwich capture principle was performed. During the course of the assay, diluted controls and diluted specimens were added to the wells and incubated. HBs specific Ab, if present, binds to the antigens. When patient serum containing HBsAg was added, it combined with the goat anti-HBsAg attached to polystyrene surface of the microwells and simultaneously bound with the horse radish peroxidase conjugated monoclonal anti-HBsAg. Wells were washed and a colorless enzyme substrate (H2O2) and chromogen (TMB, tetramethylbenzidine) were added. The enzyme acts on substrate/chromogen and produced a blue colored end product. This enzyme-substrate reaction was terminated by addition of sulfuric acid. The color changes to yellow that have occurred in each well are then measured spectrophotometrically at a wavelength of 450nm/630 nm. The yellow color intensity was directly related to concentration of Hepatitis B surface antigen in the patient sample. The sample was considered negative when OD value of the sample was less than the value of positive control.
An indirect antibody EIA assay for HBc ELISA was performed for the simultaneous detection of antibodies of total antibodies to hepatitis B virus core in human serum or plasma. It was based upon the use of a solid phase prepared with recombinant HBc antigen. The sera to be tested and the control sera were added to the wells. If antibodies to HBc were present, they will bind to the antigen fixed on solid phase. The peroxidase-labeled antibodies to human IgG and IgM are added after a washing step. They in turn bind to the specific antibodies captures on solid phase. After removal of unbound enzymatic conjugate, the antigen-antibody complex is revealed by addition of substrate. This enzyme-substrate reaction is terminated by addition of sulfuric acid. The colour changes to yellow that have occurred in each well are then measured spectrophotometrically at a wavelength of 450 nm/630 nm. The absorbance measured for a sample allows the presence and absence of antibodies to HBc to be determined. The colour intensity is proportional to the quantity of anti HBc antibodies bound to the solid phase. The sample is considered negative when OD value of the sample is less than the value of positive control.
Microplates were coated with the native Cytomegalovirus antigens, highly purified by sucrose gradient centrifugation and inactivated. The solid phase was first treated with the diluted sample and IgG to Cytomegalovirus are captured, if present, by the antigens. The peroxidase-labeled conjugated polyclonal antibodies to human IgG are added after a washing step. They in turn bind to the specific antibodies captures on solid phase. The enzyme captured on solid phase, acting on substrate and chromogen mixture, generate an optical signal that is proportional to the amount of anti-Cytomegalovirus IgG antibodies present in sample at 450/630 nm. The sample was considered negative when OD value of the sample is less than the value of positive control.
The assay is based on the principle of IgM capture where IgM class antibodies in sample are first captured by solid phase coated with hIgM antibody. A complex composed of biotinylated CMV antigen and Streptavidin, labeled with peroxidase were added after a washing step. They in turn bind to the specific antibodies captures on solid phase. The enzyme captured on solid phase, acting on substrate and chromogen mixture, generate an optical signal that is proportional to the amount of anti-Cytomegalovirus IgM antibodies present in sample at 450/630 nm.
The sample is considered negative when OD value of the sample is less than the value of positive control.
Microplates were coated with the HTLV I & II specific synthetic immunodominant antigens, derived from gp46-I, gp46II and gp21-I. The solid phase was first treated with the sample and Anti HTLV I & II are captured, if present, by the antigens coated to the microplate. The peroxidase- labeled specific synthetic antigens derived from gp46-I, gp46-II and gp21 were added after a washing step. They in turn bind to the specific antibodies captures on solid phase. The enzyme captured on solid phase, acting on substrate and chromogen mixture, generate an optical signal that is proportional to the amount of anti HTLV I & II antibodies present in sample at 450/630 nm. The sample was considered negative when OD value of the sample is less than the value of positive control. All the samples for the 0 month, 6 month, and 1 year showed absence of infectious diseases.
A primary function of albumin is to bind and stabilize a range of small molecules and ions. In in vitro, albumin acts as a multifaceted antioxidant. Its total antioxidant activity is a composite of many individual antioxidant activities. Albumin binds fatty acids and protects them from oxidation; binds copper and keeps it from participating in oxidation reactions. Albumin leads to a consideration of the extracellular and intracellular actions of the molecule, and importantly the role of its interactions with numerous ligands or bioactive factors that influence the growth of cells in culture: these include hormones, growth factors, lipids, amino acids, metal ions, reactive oxygen and nitrogen species. The interaction of albumin with the cell in relation to these co-factors has a potential impact on metabolic and biosynthetic activity, cell proliferation and survival.
Method:
Albumin was estimated quantitatively by colorimetric based method. Albumin standards were prepared at different concentrations such as 0,0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0 g/dL. The samples were diluted by 2 folds for testing. 5 μl each of diluted standards and diluted samples were transferred into wells of a clear bottom plate. 200 μl of Bromocresol green (BCG) reagent were added and incubated for 5 min at room temperature. The reagent, Bromocresol green forms a colored complex with albumin when tested positive. The intensity of the color was measured at 620 nm which is directly proportional to the albumin concentration in the sample.
Albumin was estimated by colorimetric method at initial stage, after six months and after a year. Table 8 infers that the level of albumin is higher in the UCB+MB PL when compared to standard UCB PL and FBS.
The plates were assembled for casting gel as per manufacturer's instructions.
Add 60 μl of APS (ammonium persulfate), 2.4 μl of TEMED solution and 2.4 ml of 30% Acrylamide to 3.6 ml of SDS separating gel mix and pour the gel solution between then plates till the level is 2 cm below the top edge of notched plate. Add 200 to 250 μl of water to make the surface even. After the gel are set (approximately 30-40 min), wash the top of the separating gel with distilled water and drain off the water completely. Add 40 μl of APS, 4 μl of TEMED solution and 0.67 ml of 30% Acrylamide to 3.4 ml of stacking gel mix and pour directly onto the polymerized separating gel. Insert the comb into the gel solution carefully without trapping any bubbles, about 1 cm above the separating gel. The stacking gel will set in approximately 20 min.
Take 2, 3, 4, 5 μl of provided Protein sample in 4 microfuge vials. Label them as 1, 2, 3, 4 respectively. Add 20 μl of sample loading buffer to protein samples. Place the samples in water bath for 5 min. After the stacking gel has set, carefully remove the comb and the bottom spacer. Wash the wells immediately with distil led water to remove non-polymerized acrylamide. Assemble the gel set in the Gel running apparatus as per manufacturer's instruction. Fill the top and bottom reservoir with 1×Gel running buffer. Load 20 μl protein markers in the first well and then load prepared Protein samples in well 2,3,4,5. Note down the order of loading. Connect the cords to the power supply according to the convention. Set voltage at 1OOV and switch on the power supply. When the dye front comes to 0.5 cm above the bottom of the gel, turn off the power. This will take approximately 1 to 1:30 hours. Remove the gel plates and gently open the plates apart using a spatula or similar tool, don't try to separate the plates at the notch as it might damage the notch.
Transfer the gel to a tray containing water; wash the gel for 1-2 minutes at room temperature. Decant water, cut the gel along lane 4. Transfer lanes 1-5, i.e. protein sample in 10 ml of blotting buffer taken in a Petri dish. Keep at room temperature for 10 minutes. Following incubation, proceed for electroblotting. To the gel piece add minimum of 20 ml water. Wash the gel by rotating gently. Decant the water; add 20 ml of Ezee Blue Stain. Stain at room temperature for 1-2 hours. For uniform staining and washing, place the tray on a rocker intermittently every 10 to 15 minutes. Decant the staining solution add minimum quantity of water to cover the gel. Cover the tray and leave it overnight at room temperature.
In lane one, there is a ladder with marker ranging from 10 kDa to 220 kDa. These are used to determine the size of proteins in the gel. Each band in the ladder is a known molecular weight. The samples can be determined from these known weights.
In lane two there is standard Human Albumin protein of 49 kDa. Further lane 3, 4 and 5 shows the results of tested sample.
From the gel, it is determined that standard Human Albumin protein and FBS Protein, UCB PL Protein and UCB+MB PL Protein are relatively pure proteins.
It was found that a standard Human Albumin protein will have the same molecular weight regardless of the concentration of the tested sample as the albumin levels tested were in the range of 1.9-2.5 g/dL (Table 8). Further,
Proteins were estimated by UV spectrophotometer using Bradford protein quantification protocol. 50 μg proteins were denatured and digested by Proteomic
Grade trypsin (Sigma T6567). In brief, 50 μg of protein was dissolved in 50 mM ammonium bicarbonate buffer containing 0.1% RapiGest (Waters Corporation, Mass., USA). Proteins were reduced and alkylated by treating with 100 mM dithiothreitol for 15 min at 60 ° C. and 200mM iodoacetamide for 30 min at room temperature respectively. Denatured proteins were treated with trypsin (1:25) at 37° C. for 18 h and the reaction was stopped by addition of 0.1% formic acid. Digested peptides were desalted by using C18 Zip tips (Millipore, Billerica, Mass.) and the eluted peptides were concentrated by using vacuum concentrator. The peptides were reconstituted in 3% ACN with 0.1% formic Acid and used for mass spectrometric analysis.
Peptide digests (2.5 μg) were separated by using Accela 1250 UHPLC (Thermo Fisher Scientific) equipped with a Hypersil Gold C18-reverse phase column (150*2.1 mm, 1.9 μm). The sample was loaded onto the column with 98% of mobile phase A (100% water, 0.1% formic acid (FA)) and 2% of mobile phase B (100% ACN, 0.1% FA) at 350 μl/min flow rate. Peptides were eluted with a 45 min linear gradient of 2 to 40% mobile phase B. In case of plasma samples, the LC method was extended to 120 min with a linear gradient of 2 to 50% of mobile phase B. The column temperature was set to 40° C. and auto sampler at 8° C. All samples were analyzed on hybrid quadruple Q-Exactive Orbitrap MS. The instrument tune parameters were optimized for the better results as: spray voltage 4,200 V, capillary temperature 320° C., heater temperature 200° C., S-lens RF value 55, sheath and auxiliary gases pressure were 30 and 8 psi, respectively. The samples were acquired in positive ionization mode in data-dependent manner using a top-five method with scan range from 350-1,800 m/z. MS spectra were acquired at a resolution of 70,000 with maximum injection time (IT) of 120 ms and automatic gaincontrol (AGC) value of 1 e6 ions; MS/MS spectra were acquired at 17,500 resolution with maximum IT of 120 ms and AGC value of 1e5 ions. Precursor's selectivity was performed at an isolation width of 3 m/z, under fill ratio of 0.3%, and dynamic exclusion time of 15 s. The peptide fragmentation was performed in high energy collision induced dissociation (HCD) cell using normalized HCD at 30 eV.
Proteins were searched against UniProt reviewed human protein database by using Proteome Discoverer software with following parameters, 1% FDR. The precursor and fragment initial mass error tolerance was set to 0.05 and 0.1 Da, respectively. Search parameters also included carbamidomethylation of cysteine residues as fixed modifications and methionine oxidation as variable modification.
Umbilical Cord blood platelets with maternal blood platelets (Sample 1) showed a total of 206 proteins whereas Umbilical Cord platelets (Sample 3) showed only a total of 171 proteins and commercially available serum supplement such as FBS (Sample 2) shows a total of 93 proteins (Table 9).
Thus, it can be observed that the mixture of UCB and MB platelets resulted in an addition of 35 proteins compared to the Standard Umbilical Cord blood platelets and an addition of 113 proteins compared to commercial FBS.
Conclusively, it can be stated that the optimized combination of UCB and MB results in higher protein identification and confirmation when compared to commercial or standard serum supplements.
Masses are graphed according to their relative abundance against time (See
Thus, the mixture of UCB+MB platelet lysates gives the highest amount and number of proteins.
The list of identified proteins in the optimized (UCB+MB) PL of significant value for cell culture are as follows:
Vimentin [VIME_HUMAN] (SEQ ID NO: 1) which is an intermediate filament (IF) protein that is the predominant IF in cells of mesenchymal origin such as vascular endothelium and blood cells. It facilitates cell migration and motility by recycling internalized trailing edge integrins back to the cell surface at the leading edge.
Vascular endothelial growth factor receptor 1 [VGFR1_HUMAN] (SEQ ID NO: 2) which mediates signals for differentiation. Expression of this receptor is found in vascular endothelial cells, placental trophoblast cells and peripheral blood monocytes. This protein binds to VEGFR-A, VEGFR-B and placental growth factor and plays an important role in angiogenesis and vasculogenesis. Expression of this receptor is found in vascular endothelial cells, placental trophoblast cells and peripheral blood monocytes.
Glyceraldehyde-3-phosphate dehydrogenase [E7EUT5_HUMAN] (SEQ ID NO: 3) which is an enzyme of ˜37kDa that catalyzes the sixth step of glycolysis and thus serves to break down glucose for energy and carbon molecules. GAPDH has been implicated in several non-metabolic processes, including transcription activation, initiation of apoptosis, ER to Golgi vesicle shuttling, and fast axonal, or axoplasmic transport. In sperm, a testis-Specific isoenzyme GAPDHS is expressed. GAPDH act as an oxidoreductase, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor.
Pyruvate kinase PKM [KPYM_HUMAN] (SEQ ID NO: 4) which encodes a protein involved in glycolysis. The encoded protein is a pyruvate kinase that catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate to ADP, generating ATP and pyruvate. This protein has been shown to interact with thyroid hormone and may mediate cellular metabolic effects induced by thyroid hormones.
Mesoderm posterior protein 1 [MESP1_HUMAN] (SEQ ID NO: 5) which plays a role in the epithelialization of somatic mesoderm and in the development of cardiac mesoderm.
Isoform 3 of N-alpha-acetyltransferase 60 [NAA60_HUMAN] (SEQ ID NO: 6) which is a protein that was located on the Golgi apparatus and mainly catalyze the N-acetylation of transmembrane proteins. Acetylation is one of the most ubiquitous modifications that plays a vital role in many biological processes, such as transcriptional regulation1, protein-protein interaction, enzyme activity, protein stability, antibiotic resistance, biological rhythm.
Isoform 3 of Serine/threonine-protein kinase Chk1 [CHK1_HUMAN] (SEQ ID NO: 7) is encoded by this gene belongs to the Ser/Thr protein kinase family; checkpoint mediated cell cycle arrest in response to DNA damage or the presence of unreplicated DNA. DNA damage induces activation of Chk1, which then transduces the checkpoint signal and facilitates cell cycle arrest and DNA damage repair.
Fructose-bisphosphate aldolase A [ALDOA_HUMAN] (SEQ ID NO: 8) encodes a protein which is a glycolytic enzyme that catalyzes the reversible aldol cleavage of fructose-1,6-biphosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde-3-phosphate or glyceraldehyde, respectively.
Isoform 4 of Triosephosphate isomerase [TPIS_HUMAN] (SEQ ID NO: 9) enhances triose-phosphate isomerase activity and ubiquitin protein ligase binding.
Trinucleotide repeat-containing gene 6C protein [TNR6C_HUMAN] (SEQ ID NO: 10) is a gene that plays a role in RNA-mediated gene silencing by micro-RNAs (miRNAs). Required for miRNA-dependent translational repression of complementary mRNAs by argonaute family proteins.
Histone H4 [H4_HUMAN] (SEQ ID NO: 11) display a peak in transcription in early S phase and are ideal models for cell cycle-regulated gene expression.
Centromere protein F [CENPF_HUMAN] (SEQ ID NO: 12) is required for kinetochore function and chromosome segregation in mitosis. Regulates recycling of the plasma membrane by acting as a link between recycling vesicles and the microtubule network.
Neutrophil defensin 1 [DEF1_HUMAN] (SEQ ID NO: 13) is a part of defensins, which are a family of antimicrobial and cytotoxic peptides thought to be involved in host defense. The protein encoded by this gene, defensin, alpha 1, is found in the microbicidal granules of neutrophils and likely plays a role in phagocyte-mediated host defense.
Isoform 2 of Heat shock cognate 71 kDa protein [HSP7C_HUMAN] (SEQ ID NO: 14) binds to nascent polypeptides to facilitate correct protein folding. Its role in protein folding contributes to its function in signal transduction, apoptosis, protein homeostasis, and cell growth and differentiation.
Mannose-binding protein C [MBL2_HUMAN] (SEQ ID NO: 15) is a pattern recognition molecule of the innate immune system. This provides the host with a first-line of defense before the adaptive immune system becomes operative.
Protein S100-A8 [S10A8_HUMAN] (SEQ ID NO: 16) encodes a vitamin K-dependent plasma protein that functions as a cofactor for the anticoagulant protease, activated protein C (APC) to inhibit blood coagulation. It is found in plasma in both a free, functionally active form and also in an inactive form complexed with C4b-binding protein. Mutations in this gene result in autosomal dominant hereditary thrombophilia. An inactive pseudogene of this locus is located at an adjacent region on chromosome 3. Alternative splicing results in multiple transcript variants encoding different isoforms that may undergo similar processing to generate mature protein.
Serpin A12 [SPA12_HUMAN] (SEQ ID NO: 17) belongs to a family of serpins, which are a broadly distributed family of protease inhibitors that use a conformational change to inhibit target enzymes. They are central in controlling many important proteolytic cascades, including the mammalian coagulation pathways.
Synaptotagmin-13 [SYT13_HUMAN] (SEQ ID NO: 18) based on their brain/endocrine distribution and biochemical properties, in particular C2 domains of certain synaptotagmins bound to calcium. synaptotagmins were proposed to function as calcium sensors in the regulation of neurotransmitter release and hormone secretion.
Isoform 2 of Tubulin alpha-1B chain [TBA1B_HUMAN] (SEQ ID NO: 19) is part of microtubules, which are built from a basic α/β-tubulin building block, yet subpopulations of microtubules can be differentially marked by a number of post-translational modifications. Tubulin modifications play an important role in regulating microtubule properties, such as stability and structure, as well as microtubule-based functions, such as ciliary beating, cell division, and intracellular trafficking.)
Profilin-1 [PROF1_HUMAN] (SEQ ID NO: 20) encodes a member of the profilin family of small actin-binding proteins. The encoded protein plays an important role in actin dynamics by regulating actin polymerization in response to extracellular signals.
Adenylyl cyclase-associated protein [B4DNW7_HUMAN] (SEQ ID NO: 21) is a Receptor for Human Resistin and Mediates Inflammatory Actions of Human Monocytes.
C-myc promoter-binding protein 1 [E2DRY6_HUMAN] (SEQ ID NO: 22) encodes a DENN domain-containing protein that may function as a guanine nucleotide exchange factor that specifically activates Ras-related protein Rab-10. This protein also contains an interferon stimulated response element-binding domain and may be involved in regulating the v-myc avian myelocytomatosis viral (MYC) oncogene.)
Mitochondrial heat shock 60 kD protein 1 variant 1 [B3GQS7_HUMAN] (SEQ ID NO: 23) are generally responsible for preventing damage to proteins in response to high levels of heat. It may facilitate the correct folding of imported proteins and may also prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix.
Protein S100 [B2R4M6_HUMAN] (SEQ ID NO: 24) are a family of low-molecular-weight proteins characterized by two calcium-binding sites that have helix-loop-helix (“EF-hand type”) conformation. They are also considered as Damage-associated molecular pattern molecules (DAMPs) and knockdown of AHR down regulates the expression of S100 proteins in THP-1 cell.
Beta tropomyosin isoform [A7XZE4_HUMAN] (SEQ ID NO: 25) reduction associated with transformation, regulates anoikis. Associated with establishing focal adhesions. Restores stress fibers in transformed cells.
Peroxiredoxin-1 [A0A0A0MRQ5_HUMAN] (SEQ ID NO: 26) encodes a protein that may play an antioxidant protective role in cells and may contribute to the antiviral activity of CD8(+) T-cells. This protein may have a proliferative effect and play a role in cancer development or progression.
The sample to be tested is inoculated into the vial which is entered into the BACTEC (BD BACTE FX 400 blood culture system) instrument for incubation and periodic reading. Each vial contains a sensor which responds to the concentration of CO2 produced by the metabolism of microorganisms or the consumption of oxygen needed for the growth of microorganisms. The sensor is monitored by the instrument every ten minutes for an increase in its fluorescence, which is proportional to the increasing amount of CO2 or the decreasing amount of O2 present in the vial. A positive reading indicates the presumptive presence of viable microorganisms in the vial.
Mycoplasma contamination is detected using PCR technique. In this method, DNA extraction of given sample is carried out and PCR is performed on the samples, using primers specific for mycoplasma DNA along with dNTPs and another DNA synthesis enzyme. Running the PCR product on a gel shows the presence/absence of mycoplasma DNA by band(s) of distinct sizes.
Bacterial endotoxin level is determined using Endosafe PTs reader method. The test is a rapid, point-of-use handheld spectrophotometer that uses disposable cartridge for accurate convenient and realtime endotoxin testing, glucan identification and gram identification. All the tests showed no microbial growth and the endotoxin values showed <0.750 EU/ml in all the samples.
Product safety was performed by testing sterility, mycoplasma and endotoxin at manufacturing time, after six month and after a year to examine whether the product is free from any contaminations.
Umbilical Cord blood leukocyte cell concentrate containing Hematopoietic Stem cells (HSC) that were seeded for HSC-CFU assay in one 35 mm cell culture
Petri plates, were used. Leukocyte cell concentrate sample containing HSC (1.3 ml having 2×104 cells) were seeded in 35 mm petri plate cultured with 1.5 ml of Methocult, IMDM, growth factors and 5% FBS/5%UCB-PL/UCB-MB PL. These cultured plates were placed in 90/100 mm culture dish with one more 35 mm culture dish (without lid) containing sterile DW. These Petri plates were incubated in CO2 incubator with 5% CO2 at 37° C. under humidified conditions for 14 days of incubation.
After 14 days of incubation, 35 mm culture plates were taken out from CO2 incubator and observed under inverted microscope. After opening the lid of plates, plates were kept in 60 mm gridded scoring dish under the stage of an inverted microscope. Colonies were enumerated of each plate using manual blood cell counter. After counting the colonies photographs of the same were taken under 100× magnification. Both the colonies CFU-GM (Colony Forming Unit-Granulocyte Macrophage) and BFU-E (Burst Forming Unit- Erythrocyte) colonies were counted and number of colonies were recorded.
Results: HSC CFU Assay: Below table demonstrates the results of HSC CFU colonies—CFU-GM (Colony Forming Unit-Granulocyte Macrophage) and BFU-E (Burst Forming Unit- Erythrocyte) colonies observed under inverted microscope at 14 days of culture.
#Umbilical Cord blood leukocyte cell concentrate containing Hematopoietic Stem cells (HSC) cultured with 1.5 ml of Methocult, IMDM, growth factors and 5% FBS/5%UCB-PL/UCB-MB PL
It can be observed from Table 11 and 12 that the UCB+MB PL provides better performance in terms of the CFU and BFU values for both UCB stem cells (HSC) and UCB leukocyte cell concentrate containing HSC.
The Platelet lysate of all sources (UCB+MB) PL, UCB PL, MB PL, and FBS was added to culture medium at different concentrations of 5% and 20%. The best cell growth in terms of count, viability and characterization was found in UCBPL+MBPL group in the different cell lines was tested by performing various experiments such as cell purity, cell viability, cell count, cell characterization and cryopreservation which are enlisted as below:
Cell purity test is carried out using the ELISA technique wherein protein levels in the sample are detected and compared with standard human albumin. The values of the samples are recorded. Maximum allowed limit for this is 1 g/dL.
Cell surface antigen of specific cells can be determined by using flow cytometry. In this test cell surface antibodies of specific cells are incubated with cells followed by washing and centrifugation. Cells treated with antibodies are compared with untreated cells after acquisition of cells on flow cytometer. Based on the results, percent cell population positive for specific cell surface antigen is determined and recorded.
Cells were harvested and dissolved in 10 ml of growth medium. 20 μl of cell suspension were taken and mixed with 20 μl of trypan blue dye. Load 10 μl of cell suspension along with trypan blue dye on hemocytometer. Cells were counted in all four chambers and final cell count was calculated as per following formula.
Total cell count=B1+N2+N3+N4/4×2×104×dilution factor (Where N1, N2, N3, N4 are cell counts in four chambers)
The Trypan Blue dye exclusion test is used to determine the number of viable cells present in a cell suspension. It is based on the principle that live cells possess intact cell membranes that exclude the dye, whereas dead cells do not. A viable cell will have a clear cytoplasm whereas a nonviable cell will have a blue cytoplasm.
Platelet lysate prepared using UCB+MB platelets were aliquoted in 50 ml sterile centrifuge tubes and stored at −86° C. At different intervals of 6 months and 1 year, PL aliquots were thawed at 37° C. temperature in water bath and used as a supplement for different cell culture and expansion purpose.
Cells of different lineages were cryopreserved for a time period of six months and one year. The cryopreservation was done using cell freezing medium containing either 90% of optimized PL +10% DMSO or 90% of cord blood PL +10%
DMSO and/ or 90% of FBS +10% DMSO. Further these cryovials are subjected to process of controlled freezing using Control rate freezer and reduced temperature upto −50° C. with 1° C. per min freezing rate with the help of liquid nitrogen and after attaining temperature at −50° C., the cryovials are immediately transferred at a temperature below −150° C. in liquid nitrogen tank (under Vapour phase conditions).
Cryopreserved cells of different lineages were thawed and reprocessed at different time intervals such as 6 months and one year. In this process, cryopreserved vials were removed from Liquid nitrogen tank and immediately thawed the vials containing cells in water bath at 37° C. After thawing, the cell suspension from cryovial were transferred to growth medium (DMEM, F12 with FGF) containing 10% of optimized platelet lysate or 10% of cord blood PL or 10% FBS and centrifuged at 1200-1800 rpm for 5-10 mins. Supernatant were discarded and further suspend the cell pellet in culture medium (DMEM, F12 with FGF) containing 10% of optimized platelet lysate or 10% of cord blood PL or 10% FBS and cell count and cell viability tests were performed, and their observations were recorded in the tables below.
Mycoplasma test were performed and found negative for all cell lineages.
Table 13: samples of chondrocytes were cultured with (UCB+MB) PL, UCB PL and FBS. Cell count, cell viability was performed followed by cell characterization using Flowcytometry. It was observed that the results of cell count, cell viability and cell characterization were higher in (UCB+MB)PL when compared to UCB PL and FBS.
It can be observed that the cell viability and the cell count of chondrocytes cultured using UCB+MB PL in case of all the five samples perform better as compared to the rest of the PL samples and also FBS sample, therefore, proving its higher efficacy.
Table 14: samples of osteoblasts cells were cultured with (UCB+MB) PL, UCB PL and FBS. Cell count, cell viability was performed followed by cell characterization using Flowcytometry (Table 14). It was observed that the results of cell count, cell viability and cell characterization were higher in (UCB+MB) PL when compared to UCB PL and FBS.
It can be observed that the cell viability and the cell count of osteoblasts cultured using UCB+MB PL performs better as compared to the rest of the PL samples and also FBS sample, therefore, proving its higher efficacy.
The above tables (13and 14) showed that the optimized combination of mixing umbilical cord blood derived platelet with maternal blood derived platelet envisages the same or adequate efficacy when compared to standard cord blood platelet or the commercial FBS as a serum supplement. The optimized formulation recorded and resulted the culturing, proliferation and expansion of plurality of cell lineages the like, mesenchymal stem cells derived from human umbilical cord tissue, osteoblasts differentiated from bone marrow derived mesenchymal stem cells, cardiomyocytes differentiated from human umbilical cord tissue derived mesenchymal stem cells, chondrocytes from human cartilage biopsy and buccal biopsy derived dermal fibroblasts.
This example determines the growth kinetics, self-renewing capacity, proliferation, differentiation, expansion, cell count, cell viability and cell characterization of the respective cell lineage, potential during extensive sub culturing and following cryopreservation. Primary cultures of each cell lines were established, an aliquot was cryopreserved and thawed and reprocessed, and then the populations were sub cultured parallelly for a time period of six months to one year.
Cells derived from each lineage were assayed for their kinetics of growth and their potential in response to cell proliferation, differentiation and expansion in specific medium designed to respective area of therapeutics. The results confirmed that the resultant cell lineages, including but not limiting to, the osteogenic potential, chondrogenic potential, cardiomyocytes, dermal and/or epithelial potential was conserved throughout every passage. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or cellular differentiation and expansion when the said optimized combination of UCB and MB platelet lysate were incorporated.
Importantly, these studies demonstrate that replicative senescence of parent cell is not a state of terminal differentiation since these cells remain capable of progressing through their cell lineage. The use of optimized combination of mixing umbilical cord derived platelets and maternal blood derived platelets as a measure of biological age suggests that the said platelet lysate is intermediate between embryonic and adult tissues, and as such, may provide an in-situ source for progenitor cells throughout an adult's lifetime for various therapeutic purposes.
Table 15 and Table 16 below showed that cell count, viability and characterization using differentiation markers are better and hence, showed greater efficacy of (UCB+MB) PL over UCB PL, MB PL and FBS. The comparison has also been done by using 5% each of (UCB+MB) PL, UCB PL, MB PL and FBS for culturing versus 20% each of (UCB+MB) PL, UCB PL, MB PL and FBS for culturing the chondrocytes (Table 15) and osteoblasts (Table 16). The data provided in Tables 14 and 15 are for the 0 month of culturing the cells.
Table 16 below showed that cell count, viability and characterization using differentiation markers are better and hence, showed greater efficacy of (UCB+MB) PL over UCB PL, MB PL and FBS.
From Table 15 and 16, it can be observed that culturing of the cells is equally good even in the presence of 5% UCB+MB PL as compared to 20% UCB+MB PL. Therefore, the cells can be cultured in a lesser quantity of the UCB+MB PL, hence being economically significant.
Considering the experiments above, one can appreciate the enhanced efficacy and varied applications of UCB+MB PL over and above a simple summation of its components UCB PL and MB PL as well as over FBS.
In addition to the cell lines mentioned above, a comparative study was also performed on the three more cell lines, namely, cord tissue derived mesenchymal stem cell, bone marrow derived mesenchymal stem cell, and buccal epithelial cell. The data provided in the Tables 17-19 are with respect to culturing the cells at 20% concentration of UCB+MB PL, UCB PL, MB PL, FBS as the case may be.
In concurrence with the data for the earlier two cells lines, the cell viability and the number of cells as per the three cell lines of Tables 17-19 is higher for UCB+MB PL as compared to that of FBS, UCB PL, and MB PL.
The acute toxicological effects and the tolerability of Autologous Adult Live Cultured Osteoblasts after single dose administration via subcutaneous route to Sprague Dawley rats for 14 days post dosing observation period was determined. This study was conducted to provide information on health hazards likely to arise from acute exposure in human beings.
Study Guidelines:
Good Laboratory Practice:
This study was performed following the OECD Principles on Good Laboratory Practice (revised 1997, issued January 1998) ENV/MC/CHEM (98) 17.
The Quality Assurance Unit at PRADO has reviewed the draft study plan; inspected selected study specific critical phases and audited the raw data and draft report.
Details of the methods mentioned in the subsequent section of the study report are as per the relevant Standard Operating Procedures (SOPs) at PRADO.
Test Item Details
Test System Details:
Rat was selected as a test system for this study because it is recommended by the regulatory guidelines (enumerated above) and readily available laboratory rodent species. Moreover, Sprague Dawley rats are most widely used outbred rat in biomedical research and also these are multipurpose model used for safety and efficacy testing.
All procedures followed during conduct of the study were in accordance with the Standard Operating Procedures of the PRADO and the guidelines set by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) as published in The Gazette of India, Dec. 15, 1998 and an approved Institutional Animal Ethics Committee (IAEC) protocol No. (IAEC-18-069).
A total of 20 rats (10 males and 10 females) were selected and were allowed to acclimatize for a period of nine days prior to dose administration. During this period, animals were observed daily for clinical signs.
On the last day of acclimatization period, 10 rats (5 males and 5 females) were randomly allocated for this study as Group G3. Considering the animal ethics, the data of control group was considered for this study was from PRADO/TOX-167 study.
Autologous Adult Live Cultured Osteoblasts were provided by the Sponsor and the culture was ready to inject directly.
Sample ID Distribution
As suggested by the sponsor, the maximum feasible numbers of cells per animal were selected as a dose. The subcutaneous route of administration was selected to check the acute toxicity. Efforts have been made to select route of administration as close as possible to that proposed for clinical use.
All the animals receiving Autologous Adult Live Cultured Osteoblasts were injected with 5×106 cells as a single dose which was the maximum feasible number of cells injected per animals. Control animals received vehicle supplied by the Sponsor. The dose volume for each animal was constant, i.e. 0.4 ml/animal irrespective of the body weight of the animals.
Following observations were recorded on all animals.
After subcutaneous administration of test item, all the animals were observed carefully for treatment related clinical signs, morbidity and mortality at various intervals of 10 min, 30 min, 1 hr, 2 hrs, 4 hrs and 6 hrs post dosing on first day and once daily thereafter for 14 days. The following observations were included; changes in behavior, skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and basic observations of autonomic activity (e.g., lacrimation, piloerection, pupil size and unusual respiratory pattern).
Body weights were recorded weekly. Individual animal body weights were recorded before the test item was administered and weekly thereafter. Body weight gain was calculated for individual animal.
After completion of observation period of 14 days, all the animals were fasted overnight. The blood was collected from the retro-orbital sinus in all the animals for hematological and clinical chemistry analysis.
For hematology analysis whole blood was collected in vials containing EDTA as an anticoagulant. Following hematological determinations were carried out using Transasia XP—100 Auto Analyzer;
Blood samples were collected in vials containing heparin as an anticoagulant and plasma was separated. Following parameters were evaluated on Transasia EM —180 Auto analyzers:
At termination of the study all the surviving animals were humanely sacrificed by carbon dioxide asphyxiation. All animals in the study were subjected to a gross necropsy and the gross pathological observations both external and internal were recorded.
All the individual raw data related to mortality, clinical signs, body weights and gross pathology was summarized in terms of groups and sex and presented in tabular format. Data was analyzed by student's t-test (unpaired) using Graph Pad Prism (Version 7.0).
All original raw data, QAU audited draft study plan, the approved study plan, the QAU reviewed draft study report and a copy of the final study report along with electronic data (DVD) files of the PDF (final study plan and final study report) will be retained for 9 years from the date of approval of final report. Thereafter, the archived material will be destroyed or stored for extended period as per the consent from the Sponsor.
The objective of the study is to determine the acute toxicological effects and to check the tolerability of Autologous Adult Live Cultured Osteoblasts after single dose administration via subcutaneous route to Sprague Dawley rats for 14 days post dosing observation period.
Autologous Adult Live Cultured Osteoblasts cultured using UCB and MB Platelet Lysate (Source: 3 Individual Human Autologous Adult Live Cultured Osteoblasts (OSPCS01, OSPCS02 and OSPCS03) were administered subcutaneously as a single dose of 5×106 cells/0.4 ml/animal in a group of five male and five female rats. The animals were observed for mortality and treatment related clinical signs, if any for a period of 14 days post dosing and their body weights were recorded weekly. Necropsy and gross pathology observations were performed on all rats at termination of the study
All the animals treated with a single dose of 5×106 cells/0.4 ml of
Autologous Adult Live Cultured Osteoblasts survived till the scheduled necropsy. There were no abnormal clinical signs observed in any animal throughout the treatment period which could be related to the treatment of test item. Furthermore, no treatment related gross pathological alterations in any tissues or organs of the treated animals were observed at the time of necropsy on day 15.
The detailed gross pathological examination (both external and internal) including cranial, thoracic and abdominal cavities and their contents in all treated animals did not show any lesions of pathological significance in any of the organs when compared with the control group.
Table-A: Gross pathology observations—males and females
Based on the present study conditions and the results obtained, it can be concluded that Autologous Adult Live Cultured Osteoblasts (OSPCS01, OSPCS02 and OSPCS03) at the dose of 5×106 cells/0.4 ml/animal when administered via subcutaneous route was well tolerated and no treatment related adverse effects or mortality were observed.
The present disclosure provides a method for isolating platelets based on sedimentation methods instead of the traditionally used centrifugation methods, from the discarded human umbilical cord blood (UCB) plasma and discarded maternal blood (MB) plasma; wherein the recovery was obtained in the range of 96.29%-100%. The platelet lysate produced by said method is directed to producing non-animal source cell culture supplement as an optimized supplement as compared to FBS. Due to the human origin and processing steps of the method of the present invention, the platelet lysate so produced is xenogeneic protein free supplement. Further, the raw material used in the method of preparation in the present invention is obtained from a discarded source, for example, from a laboratory. Thus, making a cost-effective method to prepare a cell culture supplement. The platelet lysate of the present disclosure provides robustness, reliability, consistency, stability and shelf-life compared to conventionally available media supplement. The combining of human fetal source embodied in umbilical cord blood (UCB) and human adult peripheral blood embodied in maternal blood (MB) as platelet source allows for combination of proteins and growth factors that enhances the efficiency of the resultant platelet lysate. The use of maternal blood (MB), which consists of higher quantity of growth factors, proteins, hormones etc., when compared to any other adult's peripheral blood due to gestational and hemodynamic changes; which thereby makes it a potential source of selection in preparing platelet lysate. The present disclosure further advantageously identifies and provides that the umbilical cord blood and maternal blood (UCB+MB) following cryopreservation anywhere from 1 month to 12 months retains efficacy in function and is able to sustain cell viability, stability and expansion of respective cell lineage to an optimal concentration. In turn, such cell lineages obtained from such optimized formulation of the umbilical cord blood and maternal blood (UCB+MB) platelet lysate is capable of being further cryopreserved with retention of resulting cell's viability, stability and expansion post-thawing in culture for respective cell lineage to an optimal concentration. The present invention further demonstrates the advantageous use of the optimized formulation of the umbilical cord blood and maternal blood (UCB+MB) platelet lysate in various human tissue derived cells therapeutic applications, including but not limited to, mesenchymal stem cells derived from human umbilical cord tissue, osteoblasts differentiated from human bone marrow derived mesenchymal stem cells, cardiomyocytes differentiated from human umbilical cord tissue derived mesenchymal stem cells, chondrocytes from human cartilage biopsy, and buccal biopsy derived dermal fibroblasts as well as epithelial cells culture with success over and above individual sources of platelet lysates, i.e., umbilical cord blood (UCB) and/or maternal blood (MB) as well as FBS.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201921005150 | Feb 2019 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IN2020/050120 | 2/7/2020 | WO | 00 |
