Patent Application
20220213433
Understanding the role of maternal health or the safety of drugs during pregnancy on early human development is an unmet need due to the high-risk status of this patient population. Animal models to understand embryonic and fetal development or test drug safety are expensive and they often have limited translation to human disease. Currently, there is no ethically acceptable human model that adequately mimics the in vivo developmental environment in a precision based way. Specifically, the effects of new or even commonly used, but untested medications, pollutants, or other molecular compounds are of particular relevance to women of reproductive age; especially when their effects on fetal health are unknown.
In one aspect, the disclosure provides methods comprising:
(a) mixing cord blood plasma, with cross-linker and stabilizer to form a mixture; and
(b) incubating the mixture for a time and under conditions to form a three-dimensional cross-linked scaffold. In one embodiment, the method comprises pre-mixing the cord blood plasma with biological cells to form a pre-mixture, wherein the pre-mixture is mixed with the cross-linker and stabilizer. In one embodiment, the cord blood plasma comprises cord blood plasma obtained from a subject having maternal pregnancy complications such as, but not limited to Type 1, Type 2 or gestational diabetes, preeclampsia, maternal obesity, smoking, multiple gestation, or preterm labor, and/or a subject having fetal pregnancy complications such as birth defects, chromosomal or hereditary disorders or intrauterine growth disturbance. In another embodiment, the biological cells comprise normal or aberrant stem cells from any suitable source, including but not limited to inducible pluripotent stem cells (iPSC), embryonic stem cells, fetal stern cells, hematopoietic stem cells, mesenchymal stem cells, bone marrow derived stem cells, umbilical cord derived stem cells, or placenta derived stem cells.
In one embodiment, the cross-linker comprises a cross-linker selected from the group consisting of calcium chloride and thrombin, or a combination thereof. In another embodiment, the stabilizer comprises tranexamic acid. In a further embodiment, no exogenous polymer is present in the three-dimensional cross-linked scaffold.
In another aspect, the disclosure provides three-dimensional cross-linked scaffolds comprising cord blood plasma. In one embodiment, the scaffold further comprises biological cells within the scaffold. In a further embodiment, the cord blood plasma comprises cord blood plasma obtained from a subject having maternal pregnancy complications such as Type 1, Type 2 or gestational diabetes, preeclampsia, maternal obesity, smoking, multiple gestation, or preterm labor, and/or a subject having fetal pregnancy complications such as birth defects, chromosomal or hereditary disorders or intrauterine growth disturbance. In one embodiment, the biological cells comprise normal or aberrant stem cells from any suitable source, including but not limited to inducible pluripotent stem cells (iPSC), embryonic stem cells, fetal stem cells, hematopoietic stein cells, mesenchymal stem cells, bone marrow derived stem cells, umbilical cord derived stem cells, or placenta derived stem cells. In further embodiments, the scaffolds comprise a cross-linker selected from the group consisting of calcium chloride and thrombin, or a combination thereof, and/or comprise tranexamic acid as a stabilizer. In another embodiment, no exogenous polymer is present in the three-dimensional cross-linked scaffold.
In a further aspect, the disclosure provides methods for using the three-dimensional cross-linked scaffolds for any suitable purpose, including but not limited to drug screening, tissue engineering, cell differentiation, toxicology studies including reproductive toxicology/teratogenicity studies, cell fate studies based on exposure to stimuli, inherent cell abnormalities, developmental biology, developmental origins of disease, regenerative medicine, etc. In one embodiment, the methods comprise
(a) contacting the three-dimensional cross-linked scaffold with a test moiety, wherein the test moiety may include, but is not limited to a drug, toxin, hormone, cytokine, small molecule, and/or other stimulus;
(b) culturing the cells of interest within the scaffold; and
(c) determining an effect of the test moiety on the cells of interest.
As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.
As used herein, “about” means +/−5% of the recited parameter.
In a first aspect, the disclosure provides methods, comprising:
(a) mixing cord blood plasma with cross-linker and stabilizer to form a mixture; and
(b) incubating the mixture for a time and under conditions to form a three-dimensional cross-linked scaffold.
The cord blood plasma may be freshly prepared, may be thawed from frozen samples, or may be obtained via any other suitable technique. The cord blood plasma may be obtained from any suitable source including collection during or following an uncomplicated or complicated pregnancy. In various embodiments, the subject may have maternal pregnancy complications such as Type 1, Type 2 or gestational diabetes, preeclampsia, maternal obesity, smoking, multiple gestation, or preterm labor. The subject may also have fetal pregnancy complications such as birth defects, chromosomal or hereditary disorders or intrauterine growth disturbance. The three-dimensional cross-linked scaffolds can be used, for example, for drug screening, tissue engineering, cell differentiation, toxicology studies including reproductive toxicology/teratogenicity studies, cell fate studies based on exposure to stimuli, inherent cell abnormalities, developmental biology, developmental origins of disease, regenerative medicine, etc.
In one embodiment, the method comprises pre-mixing the cord blood plasma with biological cells to form a pre-mixture, wherein the pre-mixture is mixed with the cross-linker and stabilizer. The pre-mixing of cord blood plasma with biological cells to form a pre-mixture may be carried out under any suitable conditions. In one embodiment, the pre-mixing is carried out at room temperature.
Any suitable biological cells may be used as deemed appropriate for an intended use. Cord blood plasma and the resulting three-dimensional cross-linked scaffolds can be used with normal or aberrant stem cells from any suitable source, including but not limited to inducible pluripotent stem cells (iPSC), embryonic, fetal, hematopoietic, mesenchymal, bone marrow derived, umbilical cord derived, or placenta derived stem cells in order to test mechanisms of normal or abnormal biologic development or screen therapeutic compounds for efficacy or developmental toxicity. In some embodiments, the cells and cord plasma are matched (i.e.: from the same subject). They may also be unmatched plasma and biological cells, or matched or unmatched combinations of plasma and biological cells from more than one subject may be used. The mixing of cord blood plasma with biological cells to form a mixture may be carried out under any suitable conditions. In one embodiment, cord plasma and resulting scaffolds from normal (non-complicated pregnancy) and abnormal pregnancy may be used to study cellular responses following exposure to normal or abnormal circulating factors including, but not limited to nutrients, fuels, hormones, cytokines, adipokines, eicosanoids, or hormones. In another embodiment, normal or abnormal cord blood plasma with circulating or added drug compounds or small molecules can be used to test responses of normal or abnormal stem cells to potential therapeutics or toxicants under variable developmental conditions.
The biological cells may be present at any suitable concentration. In one embodiment, the cells are present at between about 20 and about 107 cells/ml, between about 203106 cells/ml, between about 104 and about 107 cells/ml, between about 104 and about 106 cells/ml, about 203 and about 105 cells/ml, or between about 105 and about 107 cells/ml. In specific embodiments, the cells are present at between about 104 and about 107 cells/ml, or between about 104 and about 106 cells/ml.
In various embodiments, the cross-linker comprises a cross-linker selected from the group consisting of calcium chloride and thrombin, or a combination thereof, and/or the stabilizer is tranexamic acid. In a specific embodiment, the cross-linker comprises calcium chloride present at a concentration of between about 0.5 mg/ml and about 10 mg/ml, between about 0.5 mg/ml and about 7.5 mg/ml, between about 0.5 mg/ml and about 5 mg/ml, between about 1 mg/ml and about 10 mg/ml, between about 1 mg/ml and about 7.5 mg/ml, between about 1 mg/ml and about 5 mg/ml, between about 1.25 mg/ml and about 10 mg/ml, between about 1.25 mg/ml and about 7.5 mg/ml, or between about 1.25 mg/ml and about 5 mg/ml in the mixture (or the resulting cross-linked scaffold). In another specific embodiment, the cross-linker comprises thrombin at a concentration of between about 0.1 mg/ml and about 5 mg/ml, between about 0.25 mg/ml and about 5 mg/ml, or between about 0.5 mg/ml and about 5 mg/ml in the mixture (or the resulting cross-linked scaffold). In one specific embodiment, the cross linker comprises calcium chloride; in another specific embodiment, the calcium chloride is present at a concentration of between about 1.25 mg/ml and about 5 mg/ml in the mixture or resulting cross-linked scaffold.
In another embodiment, the stabilizer comprises tranexamic acid present at a concentration of between about 1 mg/ml and about 5 mg/ml , between about 2 mg/ml and about 5 mg/ml, or between about 2.5 mg/ml and about 5 mg/ml, in the mixture (or the resulting cross-linked scaffold).
The plasma, crosslinker, and stabilizer may be mixed in a separate container and then aliquoted into multiple wells for cross-linking as deemed appropriate for an intended use. In various embodiments, the plasma, crosslinker and stabilizer may be aliquoted into microtiter wells (for example, 24-well, 48-well, or 96-well plates), well chambers, or capsules prior to cross-linking
Any suitable incubating conditions may be used that lead to cross-linking. In one embodiment, the cross-linking incubation is carried out at about room temperature. The incubating can be carried out for any suitable period of time to accomplish the desired amount of cross-linking. In various embodiment, the cross-linking incubating is carried out for between about 5 minutes to about 8 hours, about 5 minutes to about 6 hours, about 5 minutes to about 4 hours, about 5 minutes to about 2 hours, about 30 minutes to about 8 hours, about 30 minutes to about 6 hours, about 30 minutes to about 4 hours, about 30 minutes to about 2 hours; about 1 hour to about 8 hours, about 1 hour to about 6 hours, about 1 hour to about 4 hours, about 1 hour to about 2 hours, about 2 hours to about 8 hours, about 2 hours to about 6 hours or about 2 hours to about 4 hours.
In another embodiment, no exogenous polymer is present in the three-dimensional cross-linked scaffold, which minimizes the manipulation of the natural development microenvironment provided by the scaffolds of the disclosure. In another embodiment, one or more other polymers may be added as appropriate for an intended use, including but not limited to increasing stiffness of the scaffold. In this embodiment, three-dimensional cross-linked scaffolds can recapitulate soft or stiff tissue characteristics.
The cord blood plasma may be present in the mixture at any suitable concentration. In various embodiments, the cord blood plasma is present in the mixture at a concentration of between about 30% v/v and about 80% v/v, about 30% v/v and about 70% v/v, about 30% v/v and about 60% v/v, or between about 30% v/v and about 50% v/v.
After cross-linking, cell culture media may be added to the scaffold and the scaffolds further incubated for cell growth and any uses, including but not limited to those disclosed herein. Any cell culture medium suitable for the biological cells in the scaffold may be used. The medium may be added to the top of the scaffold, may be added through the wall of the well (i.e.: not directly on top of the 3D culture), or may be added to the scaffold in any other suitable manner.
In one non-limiting embodiment, the plasma from umbilical cord and the resulting scaffolds with biological cells may comprise adding a second population of cells to the top of the scaffold and culturing the second population of cells on the scaffold. In one non-limiting embodiment, the second population may comprise stromal cells (i.e.: mesenchymal, endothelial, immune cells including but not limited to T cells, B cells, NK cells, myeloid-derived suppressor cells and monocytes). In this embodiment, the effect on the second population of cells on cells within the scaffold (cell-cell interactions or cell-ECM production) can be tested in the presence or absence of test compounds. In these embodiments, the second population of cells can be used to recreate different tissue-specific cellular niches.
In another embodiment, post-cross-linking steps, such as adding cell culture medium, cell proliferation/differentiation, and the recited uses, may be carried out at between about room temperature and about 37° C.
In a second aspect, the disclosure provides three-dimensional cross-linked scaffolds made by the method of any embodiment or combination of embodiments of the first aspect of the disclosure.
In a third aspect, the disclosure provides three-dimensional cross-linked scaffolds comprising cord blood plasma. The cord blood plasma may be obtained from any suitable source, including but not limited to a subject that has maternal pregnancy complications such as Type 1, Type 2 or gestational diabetes, preeclampsia, maternal obesity, smoking, multiple gestation, or preterm labor. The subject may also have fetal pregnancy complications such as birth defects, chromosomal or hereditary disorders or intrauterine growth disturbance.
In one embodiment, the scaffold further comprises biological cells within the scaffold. Any suitable biological cells may be used as deemed appropriate for an intended use. In one embodiment, normal or aberrant stem cells from any suitable source can be used, including but not limited to inducible pluripotent stem cells (iPSC), embryonic, fetal, hematopoietic, mesenchymal, bone marrow derived, umbilical cord derived, or placenta derived stem cells in order to test mechanisms of normal or abnormal biologic development or screen therapeutic compounds for efficacy or developmental toxicity. In one embodiment, the biological cells comprise mesenchymal stem cells, including but not limited to human mesenchymal stem cells, including, but not limited to those obtained from umbilical cord including that from the same or other subject.
In some embodiments, the cells and cord plasma are matched (i.e.: from the same subject). They may also be unmatched plasma and biological cells, or matched or unmatched combinations of plasma and biological cells from more than one subject may be used. In this embodiment, the resulting three-dimensional cross-linked scaffolds can be used, for example, for drug screening, tissue engineering, cell differentiation, toxicology studies including reproductive toxicology/teratogenicity studies, cell fate studies based on exposure to stimuli, inherent cell abnormalities, developmental biology, developmental origins of disease, regenerative medicine, etc.
In one embodiment, the biological cells are present in the scaffold at a concentration between about 203 cells/ml and about 107 cells/ml, between about 203-106 cells/ml, between about 104 and about 107 cells/ml, between about 104 and about 106 cells/ml, about 203 and about 105 cells/ml, or between about 105 and about 107 cells/ml. In specific embodiments, the cells are present at between about 104 and about 106 cells/ml.
In one embodiment, the three-dimensional cross-linked scaffold comprises a cross-linker selected from the group consisting of calcium chloride, thrombin, or a combination thereof. In various embodiments, the three-dimensional cross-linked scaffold comprises (i) calcium chloride present at a concentration of between about 0.5 mg/ml and about 10 mg/ml, between about 0.5 mg/ml and about 7.5 mg/ml, between about 0.5 mg/ml and about 5 mg/ml, between about 1 mg/ml and about 10 mg/ml, between about 1 mg/ml and about 7.5 mg/ml, between about 1 mg/ml and about 5 mg/ml, between about 1.25 mg/ml and about 10 mg/ml, between about 1.25 mg/ml and about 7.5 mg/ml, or between about 1.25 nm/ml and about 5 mg/ml; (ii) thrombin at a concentration of between about 0.1 mg/ml and about 5 mg/ml, between about 0.25 mg/ml and about 5 mg/ml, or between about 0.5 mg/ml and about 5 mg/ml in the mixture (or the resulting cross-linked scaffold), or (iii)) combinations thereof. In one specific embodiment, the cross linker comprises calcium chloride; in another specific embodiment, the calcium chloride is present at a concentration of between about 1.25 mg/ml and about 5 mg/ml in the mixture or resulting cross-linked scaffold.
In another embodiment, the scaffold comprises a stabilizer. In one embodiment, the stabilizer comprises tranexamic acid present at a concentration of between about 1 mg/ml and about 5 mg/ml , between about 2 mg/ml and about 5 mg/ml, or between about 2.5 mg/ml and about 5 mg/ml.
In a further embodiment, no exogenous polymer is present in the three-dimensional cross-linked scaffold. In another embodiment, the cord blood plasma is present in the mixture at a concentration of between about 30% v/v and about 80% v/v, about 30% v/v and about 70% v/v, about 30% v/v and about 60% v/v, or between about 30% v/v and about 50% v/v.
In all embodiments disclosed herein, the three-dimensional cross-linked scaffold may be of any suitable thickness. In various embodiments, the three-dimensional cross-linked scaffold has a thickness of between about 100 μm and about 1000 μm, between about 100 μm and about 900 μm, between about 100 μm and about 800 μm, between about 100 μm and about 700 μm, between about 100 μm and about 600 μm, between about 100 μm and about 500 μm, between about 100 μm and about 400 μm, between about 200 μm and about 1000 μm, between about 200 μm and about 900 μm, between about 200 μm and about 800 μm, between about 200 μm and about 700 μm, between about 200 μm and about 600 μm, between about 200 μm and about 500 μm or between about 200 μm and about 400 μm.
In another embodiment, a stiffness of the scaffold ranges between about 0.25 kPa to 2 kPa, between about 0.5 kPa to about 2 kPa, between about 0.75 kPa to about 2 kPa, between about 1 kPa to about 2 kPa, between about 1.25 kPa to about 2 kPa, or between about 1.5 kPa to about 2 kPa. Stiffness can be chemically-induced, or may be modified via the cells.
In another embodiment, the three-dimensional cross-linked scaffolds comprise a porous structure with a network of interconnecting fibrinogen fibers. This embodiment aids, for example, in gas diffusion, nutrient supply, and waste removal through the 3D scaffold. In embodiments in which the scaffolds contain other biological cells, the fibers may further comprise extracellular matrix fibers secreted by the cells, including but not limited to collagen. The main regulator of porosity is the fibrinogen content, but porosity can also be modulated with the crosslinkers and other chemical-inducers or by incorporating other proteins (extracellular matrix, such as collagen, laminin, etc). In various embodiments, the porosity is between about 20 μm and about 100 μm, between about 20 μm and about 75 μm, or between about 20 μm and about 50 μm in diameter. In a specific embodiment, the porosity is between 2 μm and about 8 μm in diameter.
In a fourth aspect, the disclosure provides uses of the three-dimensional cross-linked scaffold of any embodiment of combination of embodiments disclosed herein for any suitable purpose, including but not limited drug screening, tissue engineering, cell differentiation, toxicology studies including reproductive toxicology/teratogenicity studies, cell fate studies based on exposure to stimuli, inherent cell abnormalities, developmental biology, developmental origins of disease, regenerative medicine, etc.. In one embodiment, such use may comprise
(a) contacting the three-dimensional cross-linked scaffold with a test moiety, wherein the test moiety may include, but is not limited to a drug, toxin, hormone, cytokine, small molecule, and/or other stimulus;
(b) culturing the cells of interest within and/or on top the scaffold; and
(c) determining an effect of the test moiety on the cells of interest.
As discussed above, after cross-linking, cell culture media may be added to the scaffold and the scaffolds further incubated for cell growth and any uses, including but not limited to those disclosed herein. Any cell culture medium suitable for the biological cells in the scaffold may be used. The medium may be added to the top of the scaffold, may be added through the wall of the well (i.e.: not directly on top of the 3D culture), or may be added to the scaffold in any other suitable manner.
In one embodiment, cord plasma and resulting scaffolds from normal (non-complicated pregnancy) and abnormal pregnancy may be used to study cellular responses following exposure to normal or abnormal circulating factors including, but not limited to nutrients, fuels, hormones, cytokines, adipokines, eicosanoids, or hormones. In another embodiment, normal or abnormal cord blood plasma with circulating or added drug compounds or small molecules can be used to test responses of normal or abnormal stem cells to potential therapeutics or toxicants under variable developmental conditions.
Referring to
Referring to
Referring to
Human and non-human stein cells were incorporated in or seeded on human cord plasma derived three-dimensional cross-linked scaffolds to establish applications for regenerative medicine, tissue engineering, reproductive toxicology/teratogenicity studies, developmental biology, and developmental origins of disease. Fresh and bio-banked samples were collected under oversight by the Sanford Health Institutional Review Board. Specifically, umbilical (venous) cord blood and cord tissue were collected from consenting maternal donors between the ages of 18-45 years who delivered by cesarean section (n=179 subjects). Umbilical venous blood was collected by gravity into a sterile collection bag containing citrate anti-coagulant after infant delivery and cord clamping. Plasma was separated and stored at −80° C. until used to make cross-linked fibrin matrices for iWOMB. Fresh cord tissue was rinsed in iced saline and transported in sterile saline for processing the same day. Under sterile conditions, vessels were removed and the remaining tissue was minced. Human umbilical mesenchymal stem cells (hu-MSC) were derived from the Wharton's jelly by explant method or overnight digestion in collagenase type IV followed by a secondary digestion in trypsin. Hu-MSC were expanded to 70-85% confluency, aliquoted and cryopreserved in vapor phase until use. By both explant and digestion method, isolated hu-MSC meet international standards for stem cells: adhere to plastic in standard culture conditions and have >95% expression of MSC markers CD90, CD105, and CD73 by flow cytometry with little to no expression of hematopoietic or endothelial cell markers CD45, CD19, CD31, and CD34. Thawed, hu-MSC maintain self-renewal capabilities (see previous supplemental data) and are multipotent (see
Hu-MSCs were plated on cord plasma derived three-dimensional scaffolds to evaluate cell properties (
1) Stem cell maintenance media: Alpha Modification of Eagle™'s Medium (αMEM; ThermoFisher, MT15012CV), 10% Fetal Bovine Serum (FBS; Hyclone, SH3039603FBS), 1% penicillin/streptomycin (Hyclone™, SV30010), 1% L-Glutamine (Sigma Aldrich, G7513-100 ml) with or without 250 uM Amphotericin B (Sigma Aldrich, A2942-20 ML)
2) StemPro™ Adipogenesis Differentiation media (Gibco, A10070-01)
3) StemPro™ Osteogenesis Differentiation media (Gibco, A10072-01)
4) PSC Cardiomyocyte Differentiation Media: A, B and Maturation media (Gibco, A2921201).
To establish stability and function of iWOMB three-dimensional cross-linked scaffolds for a variety of cells, non-human T3T primary mouse embryonic fibroblast (NIH/3T3 ATCC® CRL1658™ ) cells were seeded on iWOMB at varying seeding densities in stem cell maintenance media. Images were captured just after seeding and after 24 hours in culture for morphological investigation and to detect optimal seeding for cell-cell and cell-matrix interactions without die off from overcrowding.
A variety of cells, including human and non-human stein cells are supported by iWOMB three-dimensional cross-linked scaffolds. Both matched (same subject) and unmatched (different subject) hu-MSC grow well in umbilical cord plasma derived scaffolds allowing cross-over studies for precision-based developmental biology and programming applications (
Referring to
To demonstrate the usefulness of iWOMB for tissue engineering, reproductive toxicology/teratogenicity studies, developmental biology, developmental origins of disease, and regenerative medicine, hu-MSC were differentiated to cardiac, adipogenic and osteogenic lineages in three-dimensional cord plasma derived scaffolds. Cardiogenesis in iWOMB is described in detail above (
Hu-MSC in iWOMBs proliferate and undergo cardiogenic, adipogenic, osteogenic differentiation by standardized techniques. Cardiogenesis yields bi-nucleated, rod-shaped, cardiomyocyte precursors which stain positive for myosin light chain 2 (MLC2) and cardiac troponin (TNNT2) (
7) We tested the ability to isolate protein and RNA from cell-seeded iWOMBs for molecular analyses. To limit patient to patient variables, hu-MSC from one subject (98) were cultured on unmatched cord blood derived scaffolds or collagen coated 24 well plates and then differentiated in to multiple lineages as described above. On advancing days 2, 7, 14 and 21 post-differentiation (D), cells were collected by collagenase I digestion for iWOMB or trypsinization for collagen. Cells in culture were pelleted and protein was isolated by trituration in RIPA lysis and extraction buffer. Cell lysate protein was quantified by DC Protein Assay (BioRad, Hercules, Calif.). RNA electropherograms were assessed and concentrations were measured by Epoch spectrophotometer (BioTek, Winooski, Vt.). To validate RNA isolation for variable assays, hu-MSC were mixed in three-dimensional cord plasma derived scaffolds in 96-well plates at increasing seeding density of 20K, 40K, 60K, 80K and 100K cells/scaffold. Cells were pellets as previously described and RNA was isolated using RNeasym™ Micro kit (Qiagen, Geimantown, Md.). RNA integrity was assessed by electropherograms using 2100 BioAnalyzer (Agilent Technologies, Santa Clara, Calif.) and RNA concentration was measured by Epoch spectrophotometer (BioTek, Winooski, Vt.). Using 1 ug of RNA, complementary DNA (cDNA) was synthesized using iScript™ cDNA Synthesis Kit and T100 Thermal Cycler (Bio-Rad, Hercules, Calif.) via manufacturer's protocol. Quantitative PCR (qPCR) was performed by TaqMan™ approach in an ABI7500 qPCR system with Absolute Blue™ qPCR Mix (ThermoFisher, Waltham, Mass.). Beta-2-microglobulin (B2M) or Ribosomal Protein Lateral Stalk Subunit PO (RPLP0), which remain stable over the course of differentiation were used as the reference genes.
Protein and RNA can be successfully isolated from cell-seeded iWOMBs. Protein collected from cell-seeded three-dimensional cross-linked scaffolds was typically greater or equal to protein recovered from collagen coated plates. Protein and RNA concentrations reflect cell numbers including during cardiogenic, osteogenic and adipogenic differentiation (
To determine the combined contribution of cells, media and the extracellular matrix (ECM) to the developmental microenvironment (DME) within iWOMBs, we measured cytokines and growth factors collected from acellular and cell-seeded three-dimensional cord plasma-derived scaffolds and compared relative expression differences over the course of hu-MSC cardiogenesis. Hu-MSC plated to three-dimensional cord plasma derived scaffolds underwent cardiogenic differentiation and protein was collected from cells and supernatant at baseline and on differentiation day 2, 5, 7, and 10 as detailed above (
Proteins within the DME can be measured in both cell lysate and supernatant from acellular and cell-seeded iWOMB. Protein within the DME of iWOMB is dynamic over the course of differentiation. Variables affecting the DME include the cord plasma derived ECM itself (
To determine whether iWOMB is a useful tool for precision-based assays, we used umbilical cord plasma and hu-MSC from control and diabetic pregnancy to identify diabetes-related differences in proteins in the DME and/or programmed cell fate. Because diabetic pregnancy varies significantly based on underlying mechanisms, we used samples from subjects with Type 1 (T1D), Type 2 (T2D) and gestational diabetes (GDM). Using our customized antibody array as detailed above (
The data demonstrate that iWOMB is a useful tool for precision-based assays. Using various combinations of normal and abnormal cord plasma derived ECM scaffolds (DME) and stem cells as shown in
Validation experiments were done to test various applications of the iWOMB. Differentiation is performed with ease in 24-well plates (
iWOMB mixtures may be aliquoted into a wide variety of microtiter wells (for example, 24-well, 48-well, or 96-well plates), chamber slides or Beem® capsules for a wide variety of applications. Depending on the well size and volume used, the three-dimensional cross-linked scaffold has a thickness of between about 100 μm and about 1000 μm, all which support tested cells. After cross-linking, culture media suitable for the cells and application in the scaffold may be added to the mixture to support cell growth, differentiation or test exposures.
This application claims priority to U.S. Provisional Patent Application Ser. No. 62/860967 filed Jun. 13, 2019, incorporated by reference herein in its entirety.
This invention was made with government support under Grant Nos. NIH/NIGMS 5 P20 GM103548-08 and NIH/NIGMS 2 P20 GM103620-06 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/037709 | 6/15/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62860967 | Jun 2019 | US |
