Patent Application
20130157353
The present invention relates to a cell-culture-bag for use in the expansion of stem cells from a crude biopsy. The present invention also relates to a system per se for expanding stems cells from a crude biopsy.
In order to treat a whole range of tissue disorders from cardiovascular disease to bone defects, it is known to use fresh bone marrow as a source of adult stem cells (mesenchymal stromal cells/MSCs), culture expand those cells into clinically required quantities (200-800 million cells) and then use them for autologous implantation as part of tissue replacement treatment.
WO2009/139703 discloses a method of expanding cells such as MSCs in a plastic bag reactor. The method uses a purified patient tissue sample of cells which are pre-cultured in a T-flask to achieve enough MSC quantity to be transferred to the plastic bag reactor. Within the plastic bag reactor, the cells are adhered to microcarriers, and culture expanded. The pre-culturing step brings with it several drawbacks. The need for the T-flask introduces material costs. The need to perform the steps of purifying and culturing the cells in the T-flask, and then perform the steps of collecting and transferring the pre-seeded cells to the plastic bag reactor introduces manpower costs for highly-trained and thus expensive technicians. Further, the transferring step inherently carries with it the risk of contamination and makes the method poorly suited for use in the field (i.e. outside of an R&D setting).
With this background in mind, according to a first aspect, the present invention may provide a cell-culture-bag for use in the expansion of stem cells from a crude biopsy, comprising:
outer walls;
a chamber located within the walls;
a first inlet, and first and second outlets, providing fluid communication with the chamber, for connection to a perfusion apparatus; and
a filter arrangement constructed to allow passage of red blood cells and block passage of stem cells from the first outlet, and block passage of microcarriers from the second outlet.
A cell-culture-bag according to the first aspect of the present invention is structured such that it is able to internally host all the operations necessary for the expansion of stem cells from a crude biopsy. The cell-culture-bag provides a closed bioreactor environment that makes it suitable for use in the field, for example, in a hospital.
In order to allow passage of red blood cells and block passage of stem cells from the first outlet, the filter arrangement filters the fluid path from the chamber to the first outlet with a relatively fine mesh size of preferably 8-20 μm, more preferably 8-12 μm and most preferably 8-10 μm.
In order to block passage of microcarriers from the second outlet, the filter arrangement filters the fluid path from the chamber to the third outlet with a relatively coarse mesh size of preferably 50-100 μm, more preferably 50-70 μm, and most preferably 60 μm.
The cell-culture-bag may comprise a third outlet, the filter arrangement being constructed to block the passage of at least microcarriers and optionally stem cells from the third outlet.
In order to block passage of microcarriers from the third outlet, the filter arrangement filters the fluid path from the chamber to the third outlet with a relatively coarse mesh size of preferably 50-100 μm, more preferably 50-70 μm, and most preferably 60 μm. When optionally the passage of stem cells from the third outlet is also blocked, the filter arrangement filters the fluid path from the chamber to the third outlet with a relatively fine mesh size of preferably 8-20 μm, more preferably 8-12 μm and most preferably 8-10 μm.
In one embodiment, the filter arrangement comprises an inner filter bag and an outer filter bag that encloses the inner filter bag.
In a preferred embodiment, the filter arrangement comprises a filter member that is associated with a said outlet and joins to the portion of the outer wall that surrounds the said outlet so as to sealingly enclose said outlet. The filter member is preferably joined to the inside surface of the outer wall. Alternatively, it may be joined to the outside surface of the outer wall. In such a case, a further cover member is also attached to the outside surface of the outer wall sealingly encloses the filter member. The filter member may be in the form of a fabric.
Preferably, the area of the filter member is substantially greater than the (cross-sectional) area of the associated outlet. A small outlet is advantageous in terms of fluid transport, and making the area of the filter member large assists in avoiding clogging.
Preferably, the join between the filter member and the outer wall includes a protuberance that serves to create a small clearance space between the filter member and the outer wall. In one embodiment, the protuberance is formed in the outer wall by a heat sealing operation that creates the join. The small clearance space assists in preventing the filter member from becoming pressed flat against the outer wall. In this way, the filter member maintains a large active area during filtering.
Preferably, each of said outlets has a respective filter member. In other embodiments, one filter member may be associated with more than one outlet and be arranged so as to sealingly enclose said more than one outlet.
Preferably, each of said outlets and the inlet has an associated coupling for connection to a conduit of the perfusion apparatus.
Preferably, the cell-culture-bag is constructed so as to permit the partitioning of the chamber into two sub-chambers. In one embodiment, the filter member associated with one of said outlets and the filter member associated with another of said outlets are spatially arranged to leave a distinct filter-free zone along which an external clamping means may act so as to clamp opposed outer walls together, thereby partitioning the chamber into two sub-chambers. In another embodiment, the first and second outlets are positioned close to the first inlet, and the partitioning means comprises a roller system capable of continuously varying the relative volume of the two sub-chambers.
Preferably, the filter arrangement/filter members are made from a material that is less attractive for attachment to the stem cells than the microcarriers and/or non-toxic to the stem cells.
In the context of the present invention, when separate filters are both referred to as “coarse” or “fine” within the same bag, they need not be the same mesh size.
According to a second aspect, the present invention may provide a system for expanding stem cells from crude biopsy, comprising:
a cell-culture-bag according to the first aspect of the present invention; and
a perfusion apparatus connected to the first inlet, and the first and second outlets for performing seeding, expansion, harvesting and collection operations on stem cells placed within the chamber.
A system according to the second aspect of the present invention carries out the expansion of stem cells from a crude biopsy within a single bioreactor environment provided by the cell-culture-bag and thereby avoids the above-mentioned drawbacks of the method of WO2009/139703.
Preferably, the perfusion apparatus comprises a means for partitioning the chamber into two or more sub-chambers. In one embodiment, the partitioning means comprises a clamp operable to clamp opposed outer walls together, thereby partitioning the chamber into two sub-chambers. In another embodiment, the partitioning means comprises a roller device operable to pinch opposed outer walls between first and second rollers, thereby partitioning the chamber into two sub-chambers. Preferably, the rollers may be rolled in unison along the bag to adjust the relative volume of the sub-chambers. By means of the partitioning means, the effective working volume of the cell-culture-bag can be limited to only one of the sub-chambers which enables the effective concentration of stem cells in that sub-chamber to exceed a threshold at which they readily adhere to the microcarriers.
According to a third aspect, the present invention may provide a system for expanding cells from a crude biopsy, comprising:
a cell-culture-bag comprising a chamber; a first inlet, and a seeding/expansion outlet, and a collection outlet, each in fluid communication with the chamber;
a perfusion apparatus connected to the first inlet, and the seeding/expansion and collection outlets;
wherein the perfusion apparatus has a seeding mode in which the first inlet and the seeding/expansion outlet are in operative connection with a seeding circuit of the perfusion apparatus, and a seeding operation is performed on stem cells within the chamber;
wherein the perfusion apparatus has an expansion mode in which the first inlet and the seeding/expansion outlet are in operative connection with an expansion circuit of the perfusion apparatus, and an expansion operation is performed on stem cells within the chamber;
wherein the perfusion apparatus has a harvesting mode in which the first inlet is in operative connection with a harvesting section of the perfusion apparatus, and an harvesting operation is performed on stem cells within the chamber;
wherein the perfusion apparatus has a collection mode in which the collection outlet is in operative connection with a collection section of the perfusion apparatus, and harvested stem cells exit from the collection outlet.
According to a fourth aspect, the present invention may provide a system for expanding cells from a crude biopsy, comprising:
a cell-culture-bag comprising a chamber; a first inlet, and a seeding outlet, an expansion outlet, and a collection outlet, each in fluid communication with the chamber;
a perfusion apparatus connected to the first inlet, and the seeding, expansion and collection outlets;
wherein the perfusion apparatus has a seeding mode in which the first inlet and the seeding outlet are in operative connection with a seeding circuit of the perfusion apparatus, and a seeding operation is performed on stems cells within the chamber;
wherein the perfusion apparatus has an expansion mode in which the first inlet and the expansion outlet are in operative connection with an expansion circuit of the perfusion apparatus, and an expansion operation is performed on stem cells within the chamber;
wherein the perfusion apparatus has a harvesting mode in which the first inlet is in operative connection with a harvesting section of the perfusion apparatus and a harvesting operation is performed on stem cells within the chamber; and
wherein the perfusion apparatus has a collection mode in which the collection outlet is in operative connection with a collection section of the perfusion apparatus, and harvested stem cells exit from the collection outlet.
System according to the third or fourth aspects of the present invention carry out the expansion of stem cells from a crude biopsy within a single bioreactor environment provided by the cell-culture-bag and thereby avoids the above-mentioned drawbacks of the method of WO2009/139703.
According to the third or fourths aspects of the present invention, a cell-culture-bag in accordance with the first aspect of the present invention is preferably used. Alternatively, a cell-culture-bag having filtering means separate from the bag, for example, in the conduits connecting to the perfusion apparatus may be used.
Preferably, the perfusion apparatus comprises a means for partitioning the chamber into two or more sub-chambers. In one embodiment, the partitioning means comprises a clamp operable to clamp opposed outer walls together, thereby partitioning the chamber into two sub-chambers. In another embodiment, the partitioning means comprises a roller device operable to pinch opposed outer walls between first and second rollers, thereby partitioning the chamber into two sub-chambers. Preferably, the rollers may be rolled in unison along the bag to adjust the relative volume of the sub-chambers. By means of the partitioning means, the effective working volume of the cell-culture-bag can be limited to only one of the sub-chambers which enables the effective concentration of stem cells in that sub-chamber to exceed a threshold at which they readily adhere to the microcarriers.
A biopsy is a sample of cells or tissue removed from a living being. A biopsy may be an entire lump or area that is removed. When a sample of tissue of fluid is removed with a needed in such a way that cells are removed without preserving the histological architecture of the tissue cells, the procedure is called a needle aspiration biopsy, or aspirate. In the context of the present invention, the terms biopsy and aspirate are used interchangeably. A biopsy or aspirate may be of any size, depending on the amount of cells needed and the amount of cells a living being may be able to donate. Biopsies and aspirates may be further purified or modified, e.g. a filter step or gradient purification to remove certain ingredients of the biopsy or aspirate, e.g. certain cells or proteins. In some cases additional tissue other than the desired biopsy tissue is present in the biopsy or aspirate due to the technique of extracting the biopsy.
For example when obtaining a bone marrow biopsy one has to go through the skin and possible a fat layer and then through the bone tissue in order to reach the bone marrow. The bone marrow biopsy then may contain bone chips and lumps of fatty tissue or cell aggregates. The additional tissue is often removed from the biopsy; however the cellular composition of the biopsy is still the same as the tissue in the living being from which it is withdrawn. Biopsies wherein only the additional tissue is removed and the cellular composition of the biopsy is the same as the cellular composition of the living tissue are referred to as cellular or crude biopsies. For example in the case of bone marrow aspirate, the bone chips, fatty tissue or cell aggregates are removed by a course filter to make sure that the cellular composition of the aspirate is the same as the bone marrow in the living being it was extracted from. This is called a cellular or crude bone marrow aspirate. A cellular or crude bone marrow aspirate contains a mixture of cells (e.g. red blood cells, platelets, white blood cells, fat cells and Mesenchymal stem cell) which is comparable to the bone marrow as it is present in the organism. In the context of the present invention, crude or cellular biopsy means a biopsy that has the same composition of cells as it has in the organism. For example, a crude bone marrow biopsy, where only the large bone chips and fat tissue is removed but has otherwise the same cellular make up as bone marrow in the organism may be placed into the cell-culture-bag of the present invention as the starting material. No other treatment is necessary. No pre-washing or pre-culturing is needed. The present invention according to all the above aspects is preferably concerned with the expansion of adult human stem cells.
Exemplary embodiments of the present invention are hereinafter described with reference to the accompanying drawings, in which:
a), (b), (c) show side views of the fourth cell-culture-bag in use.
Throughout the following description the same or corresponding parts have been given the same or corresponding reference numerals.
Stem cells: the classical definition of a stem cell requires that it possess two properties: Self-renewal—the ability to go through numerous cycles of cell division while maintaining the undifferentiated state. Potency—the capacity to differentiate into specialized cell types. In the strictest sense, this requires stem cells to be either totipotent, pluripotent or multipotent—to be able to give rise to any mature cell type, although unipotent stem cells have also been described.
The two broad types of mammalian stem cells are: embryonic stem cells that are isolated from the inner cell mass of blastocysts, and adult stem cells that are found in adult tissues. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing specialized cells, but also maintain the normal turnover of regenerative organs, such as blood, skin, or intestinal tissues.
Totipotent (a.k.a omnipotent) stem cells can differentiate into embryonic and extraembryonic cell types. Such cells can construct a complete, viable, organism. These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into nearly all cells i.e. cells derived from any of the three germ layers.
Multipotent stem cells can differentiate into a number of cells, but only those of a closely related family of cells, usually within the germ layer that the mesenchymal stem cell originates from.
Unipotent cells can produce only one cell type, their own, but have the property of self-renewal which distinguishes them from non-stem cells (e.g. skin stem cells).
Adult stem cells refer to any cell which is found in a developed organism that has two properties: the ability to divide and create another cell like itself and also divide and create a cell more differentiated than itself. Adult stem cells can be found in children, as well as adults. Pluripotent adult stem cells are rare and generally small in number but can be found in a number of tissues including umbilical cord blood and bone marrow. Most adult stem cells are lineage-restricted (multipotent) and are generally referred to by their germ-layer or tissue origin (mesenchymal stem cell, adipose-derived stem cell, endothelial stem cell, etc.). Adult stem cell treatments have been successfully used for many years to treat leukemia and related bone/blood cancers through bone marrow transplants. Adult stem cells are also used in veterinary medicine to treat tendon and ligament injuries in horses. Adult stem cells can be obtained from the intended recipient, (an autograft) the risk of rejection is essentially non-existent in these situations.
Hematopoietic stem cells (HSCs) are non-adherent multipotent stem cells that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells). Hematopoietic stem cells are found in the bone marrow of adults. Cells can be obtained directly by removal from the hip using a needle and syringe, or from the blood following pre-treatment with cytokines, such as G-CSF (granulocyte colony-stimulating factors), that induce cells to be released from the bone marrow compartment. Other sources for clinical and scientific use include umbilical cord blood, placenta, mobilized peripheral blood. For experimental purposes, fetal liver, fetal spleen, and AGM (Aorta-gonad-mesonephros) of animals are also useful sources of HSCs.
Mesenchymal stem cells (MSCs) are of stromal origin and may differentiate into a variety of tissues. MSCs have been isolated from placenta, adipose tissue, lung, bone marrow and blood, Wharton's jelly from the umbilical cord, and teeth (perivascular niche of dental pulp and periodontal ligament). Cell types that MSCs have been shown to differentiate into in vitro or in vivo include osteoblasts, chondrocytes and adipocytes. MSC are sometimes referred to Marrow Stromal Cell or Mesenchymal Stromal Cell and have been used interchangeably.
Referring to
The seeding and expansion ports 18, 20 are separated from the chamber 14 by both filter bags 25, 27. The collection coupling 22b passes through an aperture in the outer filter bag 27, whereby the collection port 22 is separated from the chamber 14 by only the inner filter bag 25.
Referring to
The perfusion apparatus 50 further comprises an expansion medium vessel 57 containing fresh nutrient, a microcarrier suspension vessel 59 containing Cytodex microcarriers (150 μm), and a cell dissociating solution vessel 61 containing cell dissociating solution, which are each coupled in parallel to the inlet port 16. A pump 63 for pumping the media from the vessels 57, 59, 61 to the inlet port 16, a control device 65, for example, an oxygenator for saturating the medium with the required concentrations of oxygen and carbon dioxide, an oxygen control flow cell 67a, and a ph control flow cell 69b are coupled in series downstream of the vessels 57, 59, 61. A conduit or tubing 71 provides fluid communication between the flow cell 69 and the inlet port 16 where it connects with the inlet coupling 16b. The conduit 71 comprises a side conduit 71a partway therealong. The side conduit 71a comprises a filter 71b having a relatively coarse mesh size of 100 μm. The side conduit 71a is adapted to permit crude aspirate to be injected into the conduit 71 and thence to enter into the cell-culture-bag 10 via the inlet port 16. In other embodiments (not shown), the crude aspirate may be injected into the cell-culture-bag via a second inlet port. A relatively coarse mesh filter may be integrated into the bag or be located external to the bag, for example, in the conduit that attaches to the second inlet port.
The perfusion apparatus 50 further comprises an oxygen control flow cell 67b, and a ph control flow cell 69b which are coupled between the outlet ports 18, 20, 22 and the expansion medium vessel 57 and a waste medium vessel 73 arranged in parallel. Also coupled to the outlet ports 18, 20, 22 is a bio-separation device 75 operable by ultrasonic separation to concentrate an MSC suspension before pumping it by a pump 77 to a sterile clinical device 79 for subsequent treatment and use. The separation device 75 is also coupled to the waste medium vessel 73.
The perfusion apparatus further comprises a rocker device 81 to which the cell-culture-bag 10 is mounted. The rocker device 81 is capable of imparting an x-y 180° motion, as indicated by the arrows R, to the cell-culture-bag 10. The perfusion apparatus 50 further comprises a bag clamp device 83 (shown abstractly in
The above-described parts of the system are preferably controlled by the controller 55.
The operation of the system 5 in processing of MSCs from crude bone marrow aspirate and expanding them is now described with reference to
In an initial set-up mode, the perfusion apparatus 50 is operable to activate the bag clamp 83 from an inoperative position to the operative position in
Next, the perfusion apparatus 50 is switched into a seeding mode, as shown in
Next, the perfusion apparatus 50 is switched into an expansion mode as shown in
Referring to
Referring still to
Thus, the cell-culture-bag 10 is able to internally host each of the seeding, harvesting, and collection operations performed in the expansion of MSCs from crude biopsy.
The perfusion apparatus 50 can be in both the harvesting mode and collection mode simultaneously, and preferably, enters the collection mode partway through the harvesting operation.
The perfusion apparatus 50 may control the transition between one mode and the next according to parameters determined a priori. Alternatively, the perfusion apparatus 50 may take measurements to guide the transitions. In one embodiment, the perfusion apparatus comprises an optical sensor that enables the colour of the fluid substances within the sub-chamber 14a during the seeding operation to be measured. As the quantity of red blood cells decreases, so does the redness of the fluid substances. In this embodiment, the material from which the upper wall portion 12a is made must be sufficiently transparent or translucent.
In this patent specification, the system 5, apparatus 50 and bag 10 are performing a method that is more extensively disclosed in the applicant's co-pending application EP 10162688.5 filed on the 12 May 2010 and entitled “Expansion of cells in a disposable bioreactor”, which is incorporated herein by reference. The system 5 and the apparatus 50 are capable of operating according to the method steps disclosed therein.
A second cell-culture-bag 10, intended for use in the
The second cell-culture-bag 10 is disposable and comprises first and second, generally rectangular, sheets 31a, 31b of ethylene vinyl acetate (EVA) heat sealed along a first contour weld 33 inwardly spaced from the edges of the sheets 31a, 31b to define a chamber 14. A second contour weld 35 formed by heat sealing along the edges of the sheets 31a, 31b creates a looped skirt 37 between the first and second contour welds 33, 35. The second cell-culture-bag 10 further comprises a first inlet port 16 positioned on a central longitudinal axis of the bag. The first inlet port 16 comprises a first inlet coupling 16b that defines an inlet 16a providing communication between the exterior of the bag to the chamber 14. The first inlet coupling 16b is held fixedly in place between the sheets 31a, 31b by the contour welds 33, 35. The second cell-culture-bag 10 further comprises a first/seeding outlet port 18, a third/expansion outlet port 20, and a second/collection outlet port 22 positioned at the first sheet 31a on the central longitudinal axis of the bag at spaced intervals from the first inlet port 16. The ports have been positioned such that the spacing between the seeding port 18 and the expansion port 20 is much greater than that between the expansion port 20 and the collection port 22 in order to create a distinct filter-free region 23 suitable for being clamped.
Each port 18, 20, 22 comprises a coupling 18b, 20b, 22b that defines an outlet 18a, 18b, 18c. Each port is the same in structure, and, for the purpose of explanation, the seeding port 18 is shown in more detail in
Referring to
The second cell-culture-bag 10 also include some guide holes 46 punched in the skirt 37 which are intended to cooperate with guide rods (not shown) in the rocker device 81 and prevent the cell-culture-bag 10 from being loaded into the rocker device 81 in the incorrect orientation. The second cell-culture-bag 10 further comprises guide pipes 47 that are located within the loop of the skirt 37. The pipes 47 are intended to cooperate with support rods (not shown) in the rocker device 81 which retain the bag in place when it is being rocked. The support rods are able to pass through the pipes 47 via slits (not shown) cut into the sheet 31a. It will have been noticed that the pipes 47 have been arranged to be clear of the clamping region 23.
In use, the second cell-culture-bag 10 performs essentially the same function as the first cell-culture-bag 10 within the system 5. The second cell-culture-bag 10 is loaded into the rocker device 81 and the ports 16, 18, 20, 22 connected up to the perfusion apparatus 50 as shown in
The second cell-culture-bag 10 is fed with standard medium from the medium bottle. The standard medium is saturated with O2 and CO2 through the oxygenator. The filtrate exiting the cell-culture-bag 10 contains RBCs which are collected in the waste-bottle.
During perfusion the amount of RBCs in the second cell-culture-bag 10 decreased which can be seen by the decrease of the red colour. After two hours filtration, the filtrate (waste bottle) and the residue (cell-culture-bag) were visually inspected using the invert microscope. The filtrate contained a high fraction of RBCs. The amount of RBCs in the residue decreased significantly while the nucleated cells are attached to the microcarriers.
In the initial set-up mode, the perfusion apparatus 50 activates the bag clamp 83 which pinches the sheets 31a, 31b together in the clamping region 23, thereby partitioning the chamber 14 into the two sub-chambers 14a, 14b. The perfusion apparatus 50 is further operable to pump microcarriers from the microcarrier suspension vessel 59 into the sub-chamber 14a. During the set-up mode, either before or after the introduction of microcarriers into the sub-chamber 14a, the user injects by syringe crude bone marrow aspirate into the sub-chamber 14a via the side conduit 71a. The coarse mesh filter 71b serves to block the passage of large blood clots and other large tissue particulates such as bone chips and the like.
Next, the perfusion apparatus 50 is switched into a seeding mode in which the first inlet 16 and the seeding outlet port 18 are in operative connection with a seeding circuit 51 of the perfusion apparatus 50. In this mode, medium is pumped through the sub-chamber 14a and the rocker device 81 subjects the cell-culture-bag 10 to a gentle rocking motion to keep the microcarriers in suspension. In this mode, the effective working volume of the cell-culture-bag 10 is limited to only the sub-chamber 14a, which enables the effective concentration of microcarriers in the sub-chamber 14a to exceed a threshold at which the MSCs of the bone marrow aspirate readily adhere to the microcarriers. Referring to
Next, the perfusion apparatus 50 is switched into the expansion mode in which the bag clamp 83 is released, thereby restoring the chamber 14 and the full working volume of the bag, and the expansion circuit 52 is established between the first inlet port 16 and the expansion port 20. In this mode, a more vigorous rocking motion (than in the seeding mode) is required to prevent formation of microcarrier/cell aggregates. Under these conditions, the fresh nutrient being pumped from the expansion medium vessel 57 enables cell expansion to take place. The adherent MSCs are confined to the bag by the second filter member 43. Both the maintenance of a large active surface area during filtering, and the selection of the filter material which is less attractive for MSC attachment than the microcarriers serve to militate against clogging at the filter member.
Next, the perfusion apparatus 50 enters into a harvesting mode in which the first inlet port 16 is in operative connection with a harvesting section 53 of the perfusion apparatus 50. In this mode, the cell disassociating solution is pumped into the chamber 14 and the rocker device 81 subjects the cell-culture-bag 10 to a relatively vigorous rocking motion in order to facilitate detachment of the MSCs from the microcarriers. Under these conditions, the MSCs become detached from the microcarriers. Next, the perfusion apparatus 50 then enters into a collection mode in which the collection port 22 is switched into operative connection with the collection section 54 of the perfusion apparatus 50. In this mode, the detached MSCs exit from the collection port 22 through the third filter member 44 and enter the bio-separator device 75. The 60 mm mesh of the third filter member 44 confines the microcarriers, including most of those that have fragmented, to the chamber 14. In the bio-separator device 75 the MSC suspension is concentrated and then pumped into the sterile clinical device 79 (see
Thus, the cell-culture-bag 10 is able to internally host each of the seeding, harvesting, and collection operations performed in the expansion of MSCs from crude biopsy.
A third cell-culture-bag 10 (not shown) is identical in construction, possible variations in construction, and use to the second cell-culture-bag 10 except that instead of the second filter member 43 having a relatively fine mesh size of 15 μm, it has a relatively coarse mesh size of 60 μm that is intended to block the passage of microcarriers, but allows the passage of MSCs and red blood cells. Having such a relatively coarse meshed filter member guarding the expansion port 20 may be sufficient where the operation of the system 5 ensures that during the expansion operation the quantity of MSCs in the chamber 14 that are un-adhered is relatively low, i.e. less than 25% and preferably less than 10%.
The material used for the sheets 31a, 31b, the material used for the filter members 42, 43, 44 and their mesh sizes, and the structure of the ports 16, 18, 20, 22 and the variations thereon disclosed in relation to the second and third cell-culture-bag apply mutatis mutandis to the first cell-culture-bag.
As described above, the system 5 performs the seeding operation in the sub-chamber 14a and then in one discrete step increases the effective working volume of that of the whole chamber 14. In other embodiments, during the seeding operation, the effective working volume of the chamber/the volume of the sub-chamber 14a can be increased in more than one discrete step. The latter can be achieved by means of a bag clamp device having more than one pair of clamping jaws.
A fourth cell-culture-bag 10 intended for use in the
Referring to
In use, the fourth cell-culture-bag 10 performs essentially the same function as the second cell-culture-bag 10 within the system 5. The fourth cell-culture-bag 10 is loaded into the rocker device 81 (omitted in
Referring to
Next, the perfusion apparatus 50 is switched into a seeding mode in which the first inlet 16 and the seeding/expansion outlet port 19 are in operative connection with a seeding circuit 51 of the perfusion apparatus 50. In this mode, medium is pumped through the sub-chamber 14a and the rocker device 81 subjects the fourth cell-culture-bag 10 to a gentle rocking motion to keep the microcarriers in suspension. In this mode, the effective working volume of the cell-culture-bag 10 is limited to only the sub-chamber 14a, which enables the effective concentration of microcarriers in the sub-chamber 14a to exceed a threshold at which the MSCs of the bone marrow aspirate readily adhere to the microcarriers. The filter member 42 having a mesh size of 15 μm, allows the red blood cells and other small cells to pass out of the seeding/expansion port 19, but blocks the passage of MSCs that have adhered to the microcarriers.
Next, referring to
Next, the perfusion apparatus 50 enters into a harvesting mode in which the first inlet port 16 is in operative connection with a harvesting section 53 of the perfusion apparatus 50. In this mode, the cell disassociating solution is pumped into the chamber 14 and the rocker device 81 subjects the cell-culture-bag 10 to a relatively vigorous rocking motion in order to facilitate detachment of the MSCs from the microcarriers. Under these conditions, the MSCs become detached from the microcarriers. Next, the perfusion apparatus 50 then enters into a collection mode in which the collection port 22 is switched into operative connection with the collection section 54 of the perfusion apparatus 50. In this mode, the detached MSCs exit from the collection port 22 through the filter member 44 and enter the bio-separator device 75. The 60 nm mesh of the filter member 44 confines the microcarriers, including most of those that have fragmented, to the chamber 14. In the bio-separator device 75 the MSC suspension is concentrated and then pumped into the sterile clinical device 79 (see
Thus, the cell-culture-bag 10 is able to internally host each of the seeding, harvesting, and collection operations performed in the expansion of MSCs from crude biopsy.
In other embodiments, instead of rocker device, a rotation device is used to facilitate the detachment of the MSCs from the microcarriers.
Experimental Data
To demonstrate the technical significance and non-arbitrary nature of the preferred 8-20 μm mesh size of the filter arrangement at the first outlet, the following experimental data is included.
Filtering of Crude Biopsy Through a 5 μm Filter and a 15 μm Filter.
Crude bone marrow biopsy from which large particles such as bone chips and fat globules are removed is diluted in 2D human mesenchymal stem cells expansion medium comparatively to the dilution which was obtained during the filtration in a cell-culture-bag 10.
The diluted biopsy was divided over two 50 ml syringes which were applied as funnels and were connected to the 5 and the 15 micron filter houses. The cell suspension was filtrated by gravity and the filtration period was monitored. In another experiment the diluted crude-human-bone-marrow biopsy was divided over two 50 ml syringes connected to the 5 and the 15 micron filter houses. By adding manual pressure, the entire cell-suspensions were filtrated. The amount of red blood cells in the filtrate was determined by cell counting using the Burker-Turk hemocytometer. After filtration, the residue was obtained by means of back-flushing the filters with 50 ml 2D human mesenchymal stem cells expansion medium. The filtrate and the residue from both filters were cultured in 2D culture flasks. After 15 days culture the 2D culture flasks were harvested for cell counting to determine human mesenchymal stem cells loss due to filtration. As control, 828 μl of non-filtrated biopsy was re-suspended in 50 ml 2D Human mesenchymal stem cells expansion medium and cultured simultaneously.
Results and Discussion
Filtration by Gravity
The filtration period was monitored and is represented in attachment 1 and table 2:
Based on the results represented in table 2 it may be concluded that the filtration period needed to filter a crude-bone-marrow biopsy by gravity using a 15 micron filter is significantly shorter than the filtration period needed using a 5 micron filter.
After a filtration period of 00:10:51, 42 ml of the cell suspension was filtrated through the 15 micron filter. After a filtration period of 19:52:27, 35 ml of the cell suspension was filtrated through the 5 micron filter. This indicates that the filtration period using the 15 micron filter is at least 110 times faster compared to the 5 micron filter. Thus after 1 hour all of the cell suspension was filtered with the 15 micron filter while only 16 ml, which is less than one third of the total volume was filtered with the 5 micron filter. Even after 20 hours still not all of the cell suspension had passed the filter.
When using a volume of about 500 ml, which is the suitable volume to expand human mesenchymal stem cells, e.g. with the cell culture bag 10, using a 15 micron filter, the filtration period is about 2 hours to filter out approximately 90% of the red blood cells. When using a 5 micron filter, the filtration period would at least be increased to 219 hours (=at least 9 days). The relatively long filtration period would increase the process time to obtain expanded human mesenchymal stem cells for clinical applications. In addition, cell viability will most likely be negatively influenced by the relatively long filtration period. This result indicates that the 5 micron filter is unsuitable to filter a crude-human-bone-marrow biopsy.
Filtration by Adding Manual Pressure
During filtration, a relatively high pressure was needed to filter the crude human bone-marrow biopsy through the 5 micron filter. After filtration the amount of red blood cells in the filtrate was determined by diluting the filtrate 10 times with PBS. The yield of Red blood cells obtained in the filtrate was comparable for both filter, see table 3
After filtration the following 2D cultures were cultivated:
After 15 days culture the 2D culture flask were harvested for cell counting to determine human mesenchymal stem cells loss due to filtration. The results are represented in table 5.
The results represented in table 5 indicate that:
More than 50% compared to the control cells were obtained with the 15 micron filter while only 3.5% compared to control with the 5 micron filter. This result was confirmed by visual inspection. It should be noted that with a 15 micron filter integrated into the cell-culture-bag 10, the back-flush procedure is not necessary, due to the design of the cell-culture-bag. Therefore, higher human mesenchymal stem cells yield are expected during filtration in the cell-culture-bag 10.
Overall, it may be concluded that the 15 micron filter is suitable for the filtration of a crude-human-bone-marrow biopsy. The 5 micron filter is not suitable for the filtration of a crude-human-bone-marrow biopsy as it does not result in acceptable yield of viably human mesenchymal stem cells within an acceptable process time.
Filtration with an 8 Micron Filter
For the filtration of a cellular-crude-human-bone-marrow-biopsy, BD Falcon cell culture Inserts with an integrated 8 μm filter were used. The Physical specifications BD Falcon cell culture Inserts are represented in table 1:
Analyses and Pre-Treatment of the Cellular-Crude-Human-Bone-Marrow-Biopsy
Due to donor variations, the cellular-crude-human-bone-marrow-biopsy was analysed to determine the starting point regarding the total number of nucleated cells. The cellular-crude-human-bone-marrow-biopsy was pre-filtrated though a 100 μm cell-strainer to remove aggregates bigger than 100 μm (eg. fat, tissue and coagulated red blood cells). The filtrate (e.g. non coagulated nucleated cells and red blood cells) was diluted in human mesenchymal stem cells 2D expansion medium at a concentration of 2.50E06 cells/ml.
Filtration of the Cellular-Crude-Human-Bone-Marrow-Biopsy
As control to the experimental conditions, the cellular-crude-human-bone-marrow-biopsy, which was only pre-filtrated though a 100 μm cell-strainer to remove aggregates bigger than 100 μm, was cultured along with the experimental was cultured along with the experimental cultures. A fraction of the diluted cellular-crude-human-bone-marrow-biopsy was seeded directly in a 6-well-cultureplate, without filtration through an 8 μm filter. The control culture was not filtrated through an 8 μm filter thus a co-culture of nucleated cells and red blood cells was obtained. After 6 days culture, human mesenchymal stem cells attached to the culture surface of the 6-well-culture-plate, while red blood cells were maintained in suspension. By withdrawing the medium from the 6-well-culture-plate, the human mesenchymal stem cells were physically separated from the red blood cells.
During this experiment, a fraction of the diluted cellular-crude-human-bone-marrow-biopsy was filtrated through an 8 μm filter to physically separate the red blood cells from the human mesenchymal stem cells. The fraction of the diluted cellular-crude-human-bone-marrow-biopsy used for the experimental culture was equal to the fraction used for the control culture.
The materials used for the filtration were 6-well-plate-inserts with integrated 8 μm filter from BD Falcon in combination with 6-well-culture-plates. Blockage of the filter was prevented by filtering dynamically by means of a rocking plateau. The filters containing the diluted cellular-crude-human-bone-marrow-biopsy were placed in the CO2-incubator on a rocking plateau rocking at 5 rpm with an angle of 15 degree.
After filtration, the red blood cells smaller than 8 μm were obtained in the filtrate and the human mesenchymal stem cells bigger than 8 μm were retained by the filter. The residue, containing the human mesenchymal stem cells, was obtained by flushing the filter, 2, 3 and 4 times. The residue was cultured to determine the human mesenchymal stem cells yield. To determine the human mesenchymal stem cells loss in the filtrate, the filtrate was cultured simultaneously. All culture procedures and cell-culture equipment were equal for the control cultures and the experimental cultures.
After 1 hour filtration, a 6-well-culture-plate with the following set-up was incubated:
All the cultures were refreshed after 6 days culture. By refreshing the medium, the human mesenchymal stem cells from the control culture were physically separated from the red blood cells in suspension.
The 6-well-culture-plates were cultured for 11 days after which the human mesenchymal stem cells yields were determined by harvesting and counting the human mesenchymal stem cells.
Results and Discussion
After 6 days culture, all the cultures were refreshed and visual inspection was performed.
Some colonies and stretched cells were observed in the control culture although the amount was visibly lower than the human mesenchymal stem cells yield obtained from the residue of the filter, after flushing twice. Furthermore, it could be observed that a lot of red blood cells were attached to the colonies of the control culture, resulting in a relatively un-healthy cell-morphology
After 11 days culture the human mesenchymal stem cells yield was determined, see table 2.1.4:
These results indicate that:
These observations indicate that it is feasible to physically separate human mesenchymal stem cells (hMSCs) from red blood cells by means of filtration using an 8 μm, without losing hMSCs in the process. The hMSCs yield is increased significantly by isolating hMSCs from a cellular-crude-human-bone-marrow-biopsy prior to culture. The hMSCs yield obtained after flushing the filter two times was 28 fold higher compared to the control culture. In addition, a healthier cell-morphology is obtained.
The hMSCs yield can be further increased by means of a system in which it is not necessary to flush the filter, like the cell-culture-bag 10.
Expansion in Culture Bags
In this experiment expansion of cells in 2D culture flask were compared to 3D culture bags. Gas-permeable-cell-culture-bags were used for the 3D cultures on Cytodex 1 microcarriers. The gas-permeable-cell-culture-bags were placed on a rotating platform (which is comparable to a 180° rocking angle). The gas-permeable-cell-culture-bag was incubated in a CO2-incubator at 37° C. and 5% CO2.
Analyses of the Cellular-Crude-Human-Bone-Marrow-Biopsy
Due to donor variations, the cellular-crude-human-bone-marrow-biopsy was analysed to determine the starting point regarding the total number of nucleated cells. The cellular-crude-human-bone-marrow-biopsy was pre-filtrated though a 100 μm cell-strainer to remove aggregates bigger than 100 μm (e.g. fat, tissue and coagulated red blood cells). The filtrate (e.g. non coagulated nucleated cells and red blood cells) was diluted in human mesenchymal stem cells 2D expansion medium at a concentration of 2.50E06 cells/ml.
Filtration of the Cellular-Crude-Human-Bone-Marrow-Biopsy
As control to the experimental conditions, the cellular-crude-human-bone-marrow-biopsy, which was pre-filtrated though a 100 μm cell-strainer to remove aggregates bigger than 100 μm, was cultured along with the experimental cultures. A fraction of the diluted cellular-crude-human-bone-marrow-biopsy was seeded directly, without filtration through an 8 μm filter, in a T-75 culture flask for the 2D control. For the 3D control culture a fraction of the diluted cellular-crude-human-bone-marrow-biopsy was seeded directly into a gas-permeable-cell-culture-bag containing Cytodex microcarriers, without filtration through an 8 μm filter.
The control cultures were not filtrated through an 8 μm filter thus a co-culture of nucleated cells and red blood cells was obtained. After 6 days culture, human mesenchymal stem cells attached to the culture surface of the T-75 culture flask or Cytodex 1 microcarriers, while red blood cells were maintained in suspension. By withdrawing the medium from the cultures, the human mesenchymal stem cells were physically separated from the red blood cells. The fractions of the diluted cellular-crude-human-bone-marrow-biopsy used for control cultures were equal to the fractions used for the experimental cultures. All culture procedures and cell-culture equipment were equal for the control cultures and the experimental cultures.
During this experiment, a fraction of the diluted cellular-crude-human-bone-marrow-biopsy was filtrated through an 8 μm filter to physically separate the red blood cells from the mesenchymal stem cells. The materials used for the filtration were 6-well-plate-inserts with integrated 8 μm filter from BD Falcon in combination with 6-well-culture-plates. Blockage of the filter was prevented by filtering dynamically by means of a rocking plateau. The filters containing the diluted cellular-crude-human-bone-marrow-biopsy were placed in the CO2-incubator on a rocking plateau rocking at 5 rpm with an angle of 15 degree. After filtration, the red blood cells smaller than 8 μm were obtained in the filtrate and the human mesenchymal stem cells bigger than 8 μm were blocked by the filter. The residue, containing the human mesenchymal stem cells, was obtained by flushing the filter two times.
The residue was cultured in a T-75 culture flask for the 2D control and in a gas-permeable-cell-culture-bag cultured in a CO2-incubator containing Cytodex microcarriers for the 3D control.
In addition, a fraction of the diluted cellular-crude-human-bone-marrow-biopsy was used to isolate the human mesenchymal stem cells by means of centrifugation. The centrifuge works using the sedimentation principle, where the centripetal acceleration causes particles with a relatively high density (e.g. human mesenchymal stem cells) to separate out along the radial direction (the bottom of the centrifuge tube). By the same token, particles with a relatively low density (e.g. red blood cells) will tend to move to the top. By subtracting the top layer, red blood cells and human mesenchymal stem cells were psychically separated.
The remainder suspension from the bottom of the centrifuge tube was re-suspended. The obtained cell-suspension was cultured in T-75 culture flask for the 2D control and in a gas-permeable-cell-culture-bag containing Cytodex microcarriers for the 3D control.
In summary, human mesenchymal stem cells were isolated and cultured accordingly:
The following parameters were applied:
Cell attachment and growth in the cell-culture-bags were monitored by means of visual inspection.
Results and Discussion
After 6 days culture, all the cultures were refreshed and visual inspection was performed.
Visual inspection of the 2D cultures indicated that the highest human mesenchymal stem cells yield and the most viable human mesenchymal stem cells morphology were obtained in the filtrated culture. It was visible that the amount of red blood cells decreased by washing, however, the filtering procedure was clearly more effective.
Visual inspection of the 3D cultures indicated that the highest human mesenchymal stem cells yield and the most viable morphology were obtained in the filtrated culture. Cell-aggregates were formed in the control culture and the centrifuged culture, even though the amount of red blood cells was reduced by centrifugation. These observations indicate that human mesenchymal stem cells need to be isolated completely prior to 3D culture using Cytodex 1 microcarriers.
After 10 days culture, all the cultures were refreshed and visual inspection was performed.
Visual inspection of the 2D cultures indicated human mesenchymal stem cells expansion in all cultures. However, the highest human mesenchymal stem cells yield and the most viable morphology were obtained in the filtrated culture. Confirming the results obtained during experiment 1 described in section 2.1.
Visual inspection of the 3D cultures indicated expansion of cell-aggregates in the control culture and the centrifuged culture. No stretched human mesenchymal stem cells were observed in contrast to the filtrated culture were a lot of healthy stretched human mesenchymal stem cells were observed.
These observations indicate that in prior art procedures, human mesenchymal stem cells need to be isolated completely prior to 3D culture using Cytodex 1 microcarriers. By means of the standard 2D isolation procedure based on adherence this means that a 2D isolation step of 6 days is needed prior to 3D culture on Cytodex microcarriers. The isolation of mesenchymal stem cells can be obviated by using a filter between 8 and 20 μm and expansion of crude cellular biopsies is feasible to culture directly on Cytodex 1 microcarriers.
Filtration of a Crude-Human-Bone-Marrow-Biopsy Through a 40 Micron Filter.
The results described above indicated that is not feasible to physically separate human mesenchymal stem cells from red blood cells by means of filtration using a 100 μm. In addition it has been demonstrated that the human mesenchymal stem cells yield is increased significantly by isolating the human mesenchymal stem cells from a cellular-crude-human-bone-marrow-biopsy prior to culture.
Procedure
A crude-human-bone-marrow-biopsy was filtrated through a 40 micron filter. Before and after filtration, visual inspection was performed. In addition, the amount of nucleated cells in the biopsy was counted before and after filtration. This was done to indicate the efficiency of the filtration procedure using a 40 micron filter.
Results and Discussion
Before and after filtration, visual inspection was performed to visualize the cellular mixture of wanted cells (human mesenchymal stem cells) and un-wanted cells (e.g. red blood cells).
Visual inspection clearly indicates that is not feasible to physically separate human mesenchymal stem cells from red blood cells by means of filtration using a 40 μm.
These observations were confirmed by cell counting since the amount of nucleated cells before filtration was comparable to the amount of nucleated cells after filtration, see table 2.3.1.
The results obtained by cell counting clearly indicate that a negligible amount of nucleated cells were isolated after filtration through a 40 micron filter. From these results can be concluded that filtration crude-human-bone-marrow-biopsy through a 40 micron filter is not sufficient for the isolation of human mesenchymal stem cells.
The described results indicate that:
| Number | Date | Country | Kind |
|---|---|---|---|
| 10162688.5 | May 2010 | EP | regional |
| 10172939.0 | Aug 2010 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/NL2011/050322 | 5/11/2011 | WO | 00 | 11/21/2012 |
