Stem cells and decellularization of tissue matrix from cord tissue

Abstract

Methods and products obtained from the method for isolating and culturing mixed populations of stem cells, making decellularized tissue matrix, making decellularized tissue matrix infused with said mixed populations of stem cells, and methods of stem cell therapy are provided.

Claims

1. A method of culturing stem cells, said method comprising: a) obtaining a mixture of cells from umbilical cord tissue, said mixture of cells including differentiated cells, stem cells and progenitor cells; b) obtaining plasma, serum or platelet lysate that is HLA-matched and optionally gender matched to said mixture of cells and having at least 3 MEW loci that are matched to said mixture of cells; and c) culturing said mixture of cells under hypoxic (0.1-7% O.sub.2) conditions in a medium supplemented with 1-10% of said plasma, serum or platelet lysate.

2. The method of claim 1, wherein said culturing in step c) is three dimensional (3D) culturing in a decellularized tissue biomatrix.

3. The method of claim 2, wherein said biomatrix is from umbilical cord.

4. The method of claim 1, wherein said serum, plasma or platelet lysate is a pooled HLA-matched product, each pooled portion of said pooled HLA-matched product having a same at least 3 matched MEW loci.

5. The method of claim 2, wherein said biomatrix is obtained by steps consisting essentially of: i) obtaining an intact portion of umbilical cord tissue; ii) cleaning said intact portion of umbilical cord tissue with a sterilizing agent; iii) decellularizing said intact portion of umbilical cord tissue to produce a decellularized tissue; iv) cutting said decellularized tissue to a desired shape without removing surface membranes or blood vessels; and v) incubating said decellularized tissue with serum, plasma or platelet lysate in 0.1% to 5% oxygen environment to make said biomatrix; and vi) combining said biomatrix and said mixture of cells for said culturing step c.

6. The method of claim 2, wherein said mixture of cells and said biomatrix are obtained by a method comprising: i) obtaining a first umbilical cord tissue from a first newborn animal; ii) cleaning said first umbilical cord tissue with a sterilizing agent; iii) mechanically dissociating said first umbilical cord tissue to isolate said mixture of cells from said first umbilical cord tissue without separating vessels from said first umbilical cord tissue; iv) obtaining a second umbilical cord tissue from said first newborn animal or from a second newborn animal; v) cutting said second umbilical cord tissue to a desired shape without separating vessels from said second umbilical cord tissue and decellularizing said second umbilical cord tissue to produce said decellularized tissue biomatrix; vi) culturing said mixture of cells together with said biomatrix in step c) wherein said serum, plasma or platelet lysate is from said first newborn animal.

7. The method of claim 6, wherein said mixture of cells and said serum, plasma or platelet lysate are each a pooled HLA-matched product, each pooled portion of said pooled HLA-matched product having a same at least 3 matched MHC loci.

8. A method of culturing stem cells, said method comprising: a) obtaining stem cells; b) obtaining plasma, serum or platelet lysate that is HLA-matched and optionally gender matched to said stem cells and having at least 3 MHC loci that are matched to said stem cells; c) culturing said stem cells under hypoxic (0.1-7% O.sub.2) conditions in a three dimensional (3D) biomatrix comprising decellularized cord tissue plus a medium supplemented with 1-10% of said plasma, serum or platelet lysate.

9. The method of claim 8, wherein said stem cells and said serum, plasma or platelet lysate are each a pooled HLA-matched product, each pooled portion of said pooled HLA-matched product having a same at least 3 matched MHC loci.

10. The method of claim 8, wherein said biomatrix is obtained by steps consisting essentially of: i) obtaining an intact portion of umbilical cord tissue; ii) cleaning said intact portion of umbilical cord tissue with a sterilizing agent; iii) decellularizing said intact portion of umbilical cord tissue to produce a decellularized tissue; iv) cutting said decellularized tissue to a desired shape without removing surface membranes or blood vessels; v) incubating said decellularized tissue with serum, plasma or platelet lysate in 0.1% to 5% oxygen environment to produce said biomatrix; and vi) combining said biomatrix with said stem cells for said culturing step c.

11. The method of claim 8, wherein said stem cells and said biomatrix are obtained by a method comprising: i) obtaining a first umbilical cord tissue from a first newborn animal; ii) cleaning said first umbilical cord tissue with a sterilizing agent; iii) mechanically dissociating said first umbilical cord tissue to isolate said stem cells from said first umbilical cord tissue without separating vessels from said first umbilical cord tissue, wherein said stem cells are a mixture of differentiated cells, stem cells and progenitor cells; iv) obtaining a second umbilical cord tissue from said first newborn animal or from a second newborn animal; v) cutting said second umbilical cord tissue to a desired shape without separating vessels from said second umbilical cord tissue and decellularizing said second umbilical cord tissue to produce said decellularized tissue biomatrix; vi) culturing said stem cells together with said biomatrix in step c) wherein said serum, plasma or platelet lysate is from said first newborn animal.

12. The method of claim 11, wherein said stem cells and said serum, plasma or platelet lysate are each a pooled HLA-matched product, each pooled portion of said pooled HLA-matched product having a same at least 3 matched MHC loci.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows some of the products derived from each umbilical cord and their use singly or in combination with other products derived from the same umbilical cord.

(2) FIG. 2 is a schematic diagram illustrating possible combination of cord blood cells, cord blood serum/plasma or platelets lysate, cord tissue cells and cord matrices products, singly or combination thereof to treat a degenerated tissue.

(3) FIG. 3 is a schematic diagram illustrating the development process of cells.

(4) FIG. 4A-I are illustrations of mechanical dissociation of umbilical cord tissues to produce decellularized matrices. This figure shows how an intact whole umbilical cord devoid of blood is sliced, cut or comminuted before or after decellularization.

(5) FIG. 5 is another illustration of products derived from each umbilical cord.

(6) FIG. 6 Describes the process of collecting and preparing cells from a portion of the cord for freezing or 3D culturing.

(7) FIG. 7 Describes the process of pooling cord cells and cord tissues from more than one donors.

(8) TABLE 1 shows the possible combination of 100% genetically compatible serum, plasma or platelet lysate as well as cord blood and cord tissue-derived cells and matrix from newborn A before or after culturing at 37° C. in low oxygen tension and before using for research or transplanting in same patient A, or an HLA- and optionally sex-matched patient.

(9) TABLE 2 shows the possible combination of 100% genetically compatible serum, plasma or platelet lysate as well as cord blood and cord tissue-derived cells from newborn A with or without a pooled HLA- and optionally sex-matched cord tissue-derived matrix from a different newborn before or after culturing at 37° C. in low oxygen tension and before using for research or transplanting in same patient A or an HLA- and optionally sex-matched patient.

(10) TABLE 3 shows the possible combination of 100% genetically compatible cord blood and cord tissue-derived cells and matrix from newborn A cultured in the presence or absence of pooled HLA- and optionally sex-matched blood sera, plasmas or platelet lysates before or after culturing at 37° C. in low oxygen tension and before using for research or transplanting in same patient A or an HLA- and optionally sex-matched patient.

(11) TABLE 4 shows the possible combination of 100% genetically compatible cord blood and cord tissue-derived cells from newborn A cultured in the presence or absence of pooled HLA- and optionally sex-matched blood sera, plasmas or platelet lysates with or without pooled HLA- and optionally sex-matched cord tissue-derived matrix from the same pool of donors before or after culturing at 37° C. in low oxygen tension and before using for research or transplanting in same patient A, or an HLA- and optionally sex-matched patient.

(12) TABLE 5 lists various unlimiting embodiments of the invention, showing the possible mix of products of HLA- and optionally sex matched cord blood units, HLA- and optionally sex matched cord blood units cord tissue-derived cells, HLA- and optionally sex matched cord blood units blood sera, plasmas or platelet lysates and HLA- and optionally sex matched cord blood units cord tissue-derived matrices all derived from the same pool of donors before or after 3D culture at 37° C. in low oxygen tension and delivery to research or transplanting in an HLA- and optionally sex matched patient.

DETAILED DESCRIPTION OF THE INVENTION

(13) The present disclosure provides any one or more of the following embodiments, in any combination: 1) 3D culture expanded cord blood in the presence of autologous cord tissue-derived cells and plasma; 2) 3D culture expanded cord blood in the presence of autologous cord tissue-derived matrix and plasma; 3) 3D culture expanded cord blood in the presence of autologous cord tissue-derived cells, cord tissue-derived matrix and plasma; 4) cord blood sera and other products for use in storage and culturing of cells; 5) umbilical cord tissue-derived biomatrix, which is decellularized cord tissue with intact or broken vessels; 6) umbilical cord tissue-derived cells, which is a complete mixture of cells mechanically liberated from the cord tissue; 7) a mixture of 5 and 6, where the mix of cord tissue cells are reinfused back into the decellularized tissue, 8) a mixture of cord blood mixed with autologous 5 and 6, where the mix of cord tissue cells are reinfused back into the decellularized tissue.

(14) In any of the above, the cells can be amplified in culture before use, and the amplification can occur before storage or after storage (or both), and occur before combining the matrix with the cell or after such combination, or combinations thereof. Preferably, the cells are both stored and cultured with autologous or syngeneic serum and similar products, although HLA and gender matched serum and similar products can be used as well.

(15) Furthermore, the decellularized cord tissue can be used with any other stem cells, and are not limited to use with the cord tissue mixture of cells. Thus, they can be used with stem cells from cord blood, mixed cells from cord blood, adult stem cells from bone marrow, epithelia, adipose tissue, and the like. As yet another alternative, the mixture of cells can be combined with other cell types for use.

(16) Additional products may comprise the following, as seen in Tables 1 through 5: umbilical cord blood cells and umbilical cord tissue-derived cells, preserved hypoxia conditioned whole umbilical cord tissue-derived matrix cultured in 0.5%-7% or 0.5%-5% oxygen tension in the presence or absence of 100% genetically compatible cord blood plasma, 100% compatible umbilical cord tissue-derived matrix and cells cultured in 0.5%-7% oxygen tension in the presence of 100% genetically compatible cord blood plasma; 100% compatible umbilical cord tissue-derived matrix and umbilical cord blood mononuclear cells cultured in 0.5%-7% oxygen tension in the presence of 100% genetically compatible cord blood plasma, 100% compatible umbilical cord tissue-derived matrix and cells and umbilical cord blood mononuclear cells cultured in 0.5%-7% oxygen tension in the presence of 100% compatible cord blood plasma, 100% compatible umbilical cord blood mononuclear cells and umbilical cord tissue-derived cells cultured in 0.5%-7% oxygen tension in the presence or absence of cord blood plasma, and umbilical cord tissue-derived cells cultured in 0.5%-7% oxygen tension in the presence or absence of cord blood plasma. The products of this disclosure also include pooled HLA- and optionally sex matched cord tissue-derived cells, pooled HLA- and optionally sex matched sera, plasmas or platelet lysates obtained from pooled HLA- and optionally sex-matched blood units, HLA- and optionally sex matched whole umbilical cord tissue matrix obtained from pooled HLA- and optionally sex-matched umbilical cord tissues and the combination of these products before and after 3D culturing.

(17) Cord blood and cord tissue are collected and the cord blood separated from the tissue as much as possible. The cord blood is typically collected in anticoagulant supplemented bag then mononuclear cells or buffy coat are separated as much as possible by centrifugation from red blood cells and plasma using common blood processing techniques like Ficoll Hypaque density gradient centrifugation or the “closed technique” using AXP AutoExpress from Cesca Therapeutics or Sepax from Biosafe. Consequently, cord blood products are a bag of cord blood containing mononuclear cells made of hematopoietic lineage cells such as lymphocytes, monocytes, stem and progenitor cells as well as mesenchymal stromal cells.

(18) Cord blood processing also produces a bag of red blood cell concentrate and another bag of plasma. Serum can also be collected from coagulated cord blood that is centrifuged at 200-800 g for 5-20 min to sediment all cells. Serum is blood without fibrinogen. It contains salt, water, antibodies, non-clotting proteins like growth factors and antigens although some clotting proteins remain. It is used right away for culturing cells or immunotherapy or for cryostoring biological products. Plasma is the liquid constituent of blood when it is not coagulated. It contains salt, water, antibodies, proteins, clotting factors and antigens. Plasma is derived from anticoagulated blood that is centrifuged at 200-800 g for 5-20 min to remove blood cells. Plasma also has a higher level of proteins than serum because during serum manufacturing some proteins get lost during coagulation and sedimentation. Plasma is useful as a source of growth factors for cell culture and for cell and tissue storage. It is also useful to treat people suffering from burns, shock, trauma, and other medical emergencies. The proteins and antibodies in plasma are also used to create therapies for rare chronic conditions, such as autoimmune disorders and hemophilia.

(19) Platelets are non-nucleated fragments of mature megakaryocytes. There are several centrifugation protocols to collect platelets from anticoagulated blood. One example is to spin anticoagulated blood at 300 g for 5 min at 12° C. followed by collecting the supernatant and spinning at 700 g for 17 min. The resulting pellet contains platelets and some red and white blood cell impurities. Most plasma supernatant is removed and enough is left to resuspend the platelets. Platelets are essential for blood clotting however they also contain valuable growth factors useful for cell culture, proliferation and survival. To release these factors, platelets should be lysed. There are several ways to lyse platelets. One example is by repeated cycles of freezing at −80° C. for hours and thawing at 37° C. To remove platelets membranes, the lysate is spun at 4,000 rpm for 30 min at room temperature in a centrifuge and the supernatant is transferred to another sterile tube. Serum, plasma, platelets lysate can be used right away or stored, and typically will be used right away to culture and store the other products.

(20) The cord tissue is cleaned and cut into two portions, or it can be cut into portions first. The first portion is for the purpose of collecting all the cells inside the umbilical cord, and the second one for the purpose of obtaining a decellularized matrix. As one alternative, we could also use the same piece for both processes, when we mince the tissue or slice it fine enough to liberate the cells with gentle agitation. However, typically we use different portions for the two products.

(21) The sterilizing agent can be selected from those known in the field, including hydrogen peroxide, isopropyl alcohol, ethanol or CholaPrep™ from Becton Dickinson.

(22) In order to make a cell mixture, the cord is typically minced, although thin slices could be made as well, and the cord gently agitated or flow passed over the tissue to gently release cells, which are collected by centrifugation, sedimentation or filtration. If the cord is very thinly sliced or spiral sliced, the sheets can be gently agitated to release the cells, and the remaining cell free sheets can then be separated from the cells and used to make decellularized matrix.

(23) The cell mixture is combined with the autologous serum, platelets or lysate, although syngeneic or HLA and gender matched product could be used instead. It is also possible to pool these blood products for use. The combination of mixed cells and serum, platelets or lysate can either be stored by freezing, or they can be cultured before storage, either with or without the decellularized matrix, as desired.

(24) To make decellularized matrix, the cord portion is minced or cut into a desired shape either before or after decellularization, or both. Then the tissue is decellularized, using enzymatic, chemical, osmotic or mechanical means, but preferably avoiding harsh chemicals or enzymes that might be difficult to eliminate or might change the matrix in any way. A large numbers of washes ensures that the decellularized matrix is free of any reagents, and the matrix can be frozen, or freeze dried, or used right away. Further, it can be frozen with cord cell mixtures and/or serum, etc., or not, as desired. The resulting decellularized tissue product can be further cut into desired shape that allows the flexibility for future 3D culturing and/or differentiation of stem cells under various circumstances for tissue engineering or regenerative therapies.

(25) FIG. 4A is a side view of how to mechanically slice cord tissue before or after decellularization. FIG. 4B is a cross-section of the umbilical cord, illustrating an example of the cutting/paring path around the vessels. The umbilical cord is spirally sliced along an axis from the surface and at a desired depth with a blade or laser. As the paring progresses, the continuous paring path is illustrated in FIG. 4B, where the eventual cord matrix sheet has portions with vessel wall and portions without.

(26) In a Class 100 or 10,000 environment, the cord is straightened or elongated by placing a long glass or metal rod in the vessels or the cord itself or both. Next, the rod is rotated over a cutting razor or laser. Adjusting the depth of the blade or laser allows us to produce matrices of different thicknesses as the cord rotates over the blade or laser, or vice versa. Consequently, sheets of cut cord are prepared and packaged in sterile packages. As an alternate method, the cord can be embedded on e.g., agar or frozen and then spiral sliced, these processes helping to support the cord as it is cut.

(27) Alternatively, as exemplified in FIG. 4A-I, before or after decellularization, the cord can be cut along several planes to produce different cord matrix shapes. FIG. 4A and 4B depict how a cord tissue can be sliced in sheets as the cord tissue rotates over a cutting blade or knife. The cord can also be fixed then a knife or blade cuts the cord tissue while rotating around it. FIG. 4A is a lateral view of a whole intact umbilical cord devoid of blood and without removing blood vessels it is sliced with a knife, blade or laser from the surface to produce a sheet of tissue of certain depth. FIG. 4B is a cross section or transverse view of the umbilical cord showing the cutting path that spirals from the surface inward although this spiral cutting can be made in reverse where the cord can be cored or cut from the inside towards the surface. In any case the sheet of cord retains blood vessel walls or portions of blood vessel walls. Cord tissue can also be frozen or embedded before being cut. FIG. 4C is an illustration of different cutting angles along the umbilical cord. FIG. 4D-4I show examples of different cord tissue cutting angles or paths showed by dotted or dashed lines. Figure on far left shows examples of umbilical cord tissue cutting planes before or after decellularization. FIG. 4D is a random mince of tissue. FIG. 4E is a cross section. FIG. 4F is an oblique cut. FIG. 4G is a longitudinal cut. FIG. 4H is a curved cut. FIG. 41 is a cylindrical or circular cut resulting in a cord piece with or without whole vessels and with or without the original cord surface. These shapes range from a collection of small of tissue matrix particles (like a slurry) to large matrix-intact decellularized cord tissue pieces in the shape of sheet, blocks, and the like.

(28) The cell mixture can also be cultured together with the decellularized tissue. The decellularized tissue serves as a 3D scaffold the same as what the cells experienced in vivo, thus more closely mimicking actual cell growth. Likewise, the oxygen concentration is adjusted to a range between 0.1% and 7% at 37° C., like the in vivo condition. The culture medium is supplemented with the serum, plasma or platelet lysate obtained from autologous or syngeneic cord blood or from an HLA- and sex-matched blood, therefore reducing the incompatibility issue and the risk of cross contamination.

(29) It is to be noted that the isolated cells need not be cultured immediately after collection, because the need for such culturing may not have arisen yet. The isolated cell mixture or matrix can be preserved for private or public use until such time that a matching patient needs treatment. Further, blood samples can be collected from the donor over time to provide a sufficient source of serum/plasma/platelet lysates to supplement future cell culturing or transplantations.

(30) It is also to be noted that the cell mixture and decellularized tissue matrices need not be cultured immediately after collection, because the need for such products may also not have arisen yet. The isolated cell mixture can be preserved for public use until such time that an HLA- and or sex matched patient needs treatment, or it can be preserved for private use by the individual that provided the cord tissue at birth. Further, HLA- and optionally sex matched blood samples can be collected from the same HLA- and optionally sex matched donors to provide a sufficient source of serum/plasma/platelet lysates to supplement future cell culturing or transplantations. Alternatively, an individual can provide his own autologous blood product for use in culturing the mixed cells and/or matrix, since when the need arises, collection of blood will typically become practical as most often the cells will not be needed until adulthood.

Methods

(31) Intact cord is collected and sterilized by flushing cord vessels with sterile antibiotic and antimycotic containing calcium- and magnesium-free PBS. The cord surface is cleaned with regular alcohol swabs before placing in a sterile bag or container containing calcium- and magnesium-free PBS supplemented with antibiotics and antimycotics, sealed and safely placed in a 2° C. to 10° C. compartment of a temperature controlled damage resistant shipping box, shipped to the laboratory, preferably a hybrid facility and received no more than 16 hours post delivery. At the hybrid facility, private and public cord tissues are processed independently in designated areas in different ways. Cord blood is also collected and shipped to the same facility.

(32) For cryopreserving whole cord tissue, a piece of cord tissue is cut; a sterile plastic tube is inserted in the vessels to keep them distended or straight; the tissue is placed in a sterile tube filled with low glucose Dulbecco Modified Essential Medium (DMEM) or similar medium (e.g., a variety of stem cell media are commercially available) containing antibiotics and antimycotics, such as penicillin and streptomycin respectively, with or without 5-20% final concentration of serum, plasma or platelet lysate derived from cord blood of the same or different newborn. GMP grade cryoprotectant is added to a final concentration of 1%-10% before quick or slow freezing to below −120° C. then quarantined in the gas phase of liquid nitrogen until communicable disease diagnostics is clear. At this point, the tissue is transferred to long-term gas phase liquid nitrogen dewars designated for either private or public umbilical cord donors.

(33) To manufacture an acellular matrix that can be used in the future for autologous or allogeneic purposes to support the regeneration of bone, cartilage, skin, fat, muscle, retinal, lens and nervous tissue, a piece of cord tissue is straightened by inserting a thick glass stick in the cord vessels. Then the cord tissue is decellularized with 0.1% peracetic acid (PAA) for 2 hours with mechanical agitation and subsequent 15 minutes wash with phosphate buffered saline (PBS) before treating the tissue with DNAse and RNAse in calcium magnesium-free PBS at 37° C. for 1 hour. Cord tissue can also be decellularized with other chemical reagents, such as hypotonic and hypertonic solutions, alcohols, SDS or other ionic or non-ionic detergents, trypsin, and the like. Mechanical dissociation methods can also be used, such as convection flow, sonication, and the like. Decellularized matrix can be derived from a portion of cord tissue that was previously cut into desired shape with a blade or laser, or triturated, or homogenized followed by decellularization with or without inserting a stick in the vessels. Therefore, acellular matrix sizes range from small particles to various sizes and shapes of cord tissue, including a natural shape, sheets, blocks, and the like.

(34) Cord tissue matrix is then washed with calcium magnesium-free PBS and preserved by placing it low temperature resistant membranes or containers containing calcium magnesium-free PBS or medium supplemented or not with antibiotics and antimycotics, 1%-10% cryoprotection solution, plus 1-10% of autologous, syngeneic or HLA- and sex-matched serum, plasma or platelet lysate. The matrix and serum combination is then slowly and gradually or fast snap frozen to −20° C. or below.

(35) In contrast, when the cells are to be retained, the tissue can be finely sliced, diced or homogenized without removing surface membrane or vessels and the cells gently liberated. For example, the tissue can be completely sliced into thin strips (shaved slices), or only striated partway through, and gentle rocking or convection flow applied, if needed, to free the cells from the thin piece of tissue. Cells in solution are collected and separated from tissue matrix by filtration, sedimentation or density gradient centrifugation. Cells can also be dissociated by homogenizing whole cord tissue using a mortar and pestle or a commercially available device. This method preserves the cells for subsequent use.

(36) To isolate novel mesenchymal stem cells (along with progenitor and differentiated cells and other not yet characterized cells like non-adherent or not yet surface adhering cells), cord tissue is mechanically dissociated or cut within 16 hours of delivery into thin filaments (including vessel walls) using a sterile scalpel or homogenizer and then liberated single and clumped cells still attached to some cord tissue matrix are directly frozen slowly or quickly to below −120° C. in medium containing 1%-10% cryoprotectant preferably supplemented with serum, plasma or platelet lysate derived from the same newborn. Alternatively, isolated single cells and clumps of cells still attached to some cord tissue matrix are placed in suspension to be cultured for days or months in a prior art 3D chamber, preferably in 0.5%-7% oxygen pressure, humidified CO.sub.2 and N.sub.2 gas environment and a low glucose DMEM medium, preferably supplemented with either serum, plasma or platelet lysate derived from the same newborn. The cells can also be cultured for days or months on a cord tissue-derived biomatrix having a certain desired 3D shape. Blood components can also be isolated from the same person or animal later in life, thus maintaining a source of autologous blood components as long as possible.

(37) Although there exists a controversy over the expression of “mesenchymal stem cells” markers, the isolated cord tissue-derived cells contains a pool of cells with the following surface markers when grown in 2-dimensional culture in the presence of fetal bovine serum: CD10.sup.30, CD29.sup.+++, CD44.sup.+++, CD73.sup.++, CD54.sup.+, CD58.sup.+, CD105.sup.+, low CD106.sup.+, CD146.sup.+, CD166.sup.+, low HLA-ABC.sup.+, STRO-1.sup.+. Gatta V. et. al., (2013) Gene expression modifications in Wharton's Jelly mesenchymal stem cells promoted by prolonged in vitro culturing. BMC Genomics 2013, 14:635; Arutyunyan I. et. al., (2016) Umbilical Cord as Prospective Source for Mesenchymal Stem Cell-Based Therapy. Stem Cells International Article ID 6901286.

(38) The different plus signs define the degree of a marker's expression whereby one plus sign means little expression and more plus signs mean higher expression. Another isolated pool of cord tissue-derived cells do not have the following surface markers when grown in 2-dimensional culture in the presence of fetal bovine serum: CD3.sup.−, CD7.sup.−, CD19.sup.−, CD14.sup.−, CD28.sup.−, CD31.sup.−, CD33.sup.−, CD34.sup.−, CD38.sup.−, CD40.sup.−, CD45.sup.−, CD56.sup.−, CD62L.sup.−, CD62P.sup.−, CD80.sup.−, CD86.sup.−, CD90.sup.−, CD106.sup.−, CD117.sup.−, CD133.sup.−, CD135.sup.−, CD144.sup.−, CD271.sup.−, CD326.sup.−, HLA-DR.sup.−, Lin C. S., Ning H., Lin G., Lue T. F. (2012) Is CD34 truly a negative marker for mesenchymal stromal cells? Cytotherapy 14:1159-1163. Imran Ullah I. et. al., (2015) Human mesenchymal stem cells—current trends and future prospective. Bioscience Reports 35/art:e00191. Still another pool of isolated cord tissue-derived cells has the following markers: CD31, CD34, CD105, CD146, VE-Cadherin, VEGFR1, VEGFR2, Tie2, CXCR4, Von Willebrand, Aldehyde dehydrogenase but does not express CD1, CD115, CD45 and CD133. However, with our cell culture methods we expect these cells to have a different molecular marker signature and/or strength of marker expression. These surface markers can be used to further characterize and/or isolate cells of various differentiation potentials when cultured in 3D and/or in the presence of 0.5%-7% (or preferably 0.5%-5%) oxygen and/or in the presence of serum, plasma or platelet lysate derived from an HLA- and optionally sex-matched animal.

(39) The cord blood, tissue cells and tissues biomatrix may be pooled from more than one donors, as long as the donors are HLA- and optionally sex-matched. The process is shown in FIG. 7 that shows an example of umbilical cord blood, cord tissue cells and cord tissue matrix products pooled from three HLA- and optionally sex-matching umbilical cords. In Step 701, three umbilical cord from three HLA-matching and optionally sex-matching donor animals are obtained. Each cord is then cut into two portions, and the cord blood from each cord is processed. Anticoagulated blood is first collected from each cord then processed into serum or plasma and blood stem cell bag products. From each cord blood processed, collect: one red blood cell (RBC) bag product; one plasma bag product; one plasma- & RBC-reduced cord blood bag product, and then pool each HLA- and optionally sex-matched product type into one bag.

(40) In Step 703, portions for deriving cells are pooled together, by mechanical dissociation of tissue to extract all cells from the first cord portion. Example of pooled HLA-and/or sex-matched cord tissue-derived cells product is shown, and optionally being frozen in sterile vials.

(41) In step 705, portions for deriving matrices are pooled together, by extracting biomatrix from the second cord portion. Example of Pooled HLA- and/or sex matched 3D decellularized cord tissue biomatrix product that can be further cut into desired shape, and optionally frozen in sterile container.

(42) In some instances where a patient has not saved his or her umbilical cord, or when a newborn is affected by a genetic disease, it is best to treat that individual with cell and matrix products derived from HLA matching and preferably sex matching donors. The greater the HLA and sex match the greater the chance of engraftment and tolerance of transplanted cells, matrices and tissues.

(43) Before or after culture, HLA- and optionally sex-matched cells and matrices cultured alone or in combination are collected and a sample is characterized by some or all of the following: sterility, size, cell surface and intracellular markers, cell proliferation rate, cell self-renewal and differentiation potential, matrix strength and cell infiltration, using microscopy, flow cytometry, cell and molecular biology techniques, as well as engraftment and regeneration assays in animal models. Concurrently, all cells are collected, washed and suspended in fresh medium containing HLA and/or sex matched cord blood serum, plasma or platelet lysate and 1% to 10% GMP grade cryoprotectant before slow and gradual or fast freezing to −120° C. or below, then quarantined in the gas phase of liquid nitrogen until communicable disease diagnostics is clear. Until then, the cells or tissues are transferred to long-term gas phase liquid nitrogen dewars designated for either private or public umbilical cord donors.

(44) On use, the cells can be allowed to attach or are reinfused back in the decellularized tissue biomatrix of a desired shape. This is done by combining the two using varying concentration of cells and decellularized tissue biomatrices and allowing the cells to attach and diffuse into the matrix, or by injecting them thereinto, depending on matrix shape and size. The cells can be used alone, but the decellularized tissue matrix provides a useful 3D scaffold and a regulated environment for stem cell maintenance and growth, and is a preferred methodology. Umbilical cord derived cells and decellularized tissue matrix can be used alone or in any combinations for animal or human therapeutic purposes. See FIG. 1, FIG. 2, Table 1, Table 2, Table 3, Table 4, and Table 5.

(45) Although various embodiments of the method and apparatus of the present disclosure have been illustrated in the accompanying Drawings (FIGS. 1 through 7 and Tables 1 through 5) and described in the foregoing Detailed Description, it will be understood that the disclosure is not limited to the embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the spirit of the disclosure as set forth herein.

(46) The following are each incorporated by reference herein in its entirety for all purposes: Gerlach J C, et al., Dynamic 3D culture promotes spontaneous embryonic stem cell differentiation in vitro, Tissue Eng Part C Methods. 2010 Feb;16(1):115-21. Bosch J, et al., Distinct Differentiation Potential of “MSC” Derived from Cord Blood and Umbilical Cord: Are Cord-Derived Cells True Mesenchymal Stromal Cells?, Stem Cells and Development (2012). U.S. application Ser. No. 13/890,134, filed May 8, 2013, and 61/644,423, filed May 8, 2012. U.S. Pat. No. 8,656,670 Facilities for hybrid tissue banks Akkermann R., Beyer F., Küry P. (2017) “Heterogeneous populations of neural stem cells contribute to myelin repair”, Neural Regen Res. Apr;12(4):509-517. Arutyunyan I. et. al., (2016) Umbilical Cord as Prospective Source for Mesenchymal Stem Cell-Based Therapy. Stem Cells International Article ID 6901286. Badylak Steven (2014) Decellularized Allogeneic and Xenogeneic Tissue as a Bioscaffold for Regenerative Medicine: Factors that Influence the Host Response. Annals of Biomedical Engineering, Vol. 42, No. 7. DeLima M. et. al., (2012) Cord-Blood Engraftment with Ex Vivo Mesenchymal-Cell Coculture, NEJM 367;24. De Waele M. et. al., (2004) Growth factor receptor profile of CD34+ cells in normal bone marrow, cord blood and mobilized peripheral blood. Eur J Haematol. Mar;72(3):193-202. Emnett R. J. et. al., (2016) Evaluation of Tissue Homogenization to Support the Generation of GMP-Compliant Mesenchymal Stromal Cells from the Umbilical Cord. Stem Cells International. Article ID 3274054. Friedman R. et. al., (2007) Umbilical Cord Mesenchymal Stem Cells: Adjuvants for Human Cell Transplantation. Biology of Blood and Marrow Transplantation 13:1477-1486. Gatta V. et. al., (2013) Gene expression modifications in Wharton's Jelly mesenchymal stem cells promoted by prolonged in vitro culturing. BMC Genomics 2013, 14:635. Gentile P. et. al., (2017) Concise Review: The Use of Adipose-Derived Stromal Vascular Fraction Cells and Platelet Rich Plasma in Regenerative Plastic Surgery. Stem Cells;35:117-134. Gharibi B., Hughes F. J., (2012) Effects of Medium Supplements on Proliferation, Differentiation Potential, and In Vitro Expansion of Mesenchymal Stem Cells, Stem Cells Translational Medicine, 1:771-782. Imran Ullah I. et. al., (2015) Human mesenchymal stem cells—current trends and future prospective. Bioscience Reports 35/art:e00191. Jung J, Moon N, Ahn J Y, et al. Mesenchymal stromal cells expanded in human allogenic cord blood serum display higher self-renewal and enhanced osteogenic potential. Stem Cells and Development. 2009;18(4):559-571. Keating, A. (2012) “Mesenchymal Stromal Cells: New Directions”, Cell Stem Cell 10, 709-716. Lin C. S., Ning H., Lin G., Lue T. F. (2012) Is CD34 truly a negative marker for mesenchymal stromal cells? Cytotherapy 14:1159-1163. Lo Sardo, V. et al. (2017) Influence of donor age on induced pluripotent stem cells. Nat. Biotechnol. 35,69-74. McNiece I, Gluckman E, Wagner J E, et al. Ex vivo expansion of umbilical cord blood hemopoietic stem and progenitor cells. Exp Hematol. 2004;32:409-413. Mendicino M., Bailey A. M., Wonnacott K., Puri R. K., Bauer S. R., (2014) “MSC-Based Product Characterization for Clinical Trials: An FDA Perspective”, Cell Stem Cell 14, Feb 6. Meral Beksac (2016) How to Improve Cord Blood Transplantation. By Enhancing Cell Count or Engraftment? Frontiers in Medicine, Vol. 3, Article 20. Phadnis S M, Joglekar M V, Venkateshan V, Ghaskadbi S M, Hardikar A A, Bhonde R R. Human umbilical cord blood serum promotes growth, proliferation, as well as differentiation of human bone marrow-derived progenitor cells. In Vitro Cellular and Developmental Biology-Animal. 2006;42(10):283-286 Shakouri-Motlagh A. et. al., (2017) Native and solubilized decellularized extracellular matrix: A critical assessment of their potential for improving the expansion of mesenchymal stem cells. Acta Biomaterialia 55 (2017) 1-12. Sharpless N. E. and De Pinho R. A., (2007) “How stem cells age and why this makes us grow old”, Nature Rev. Mol. Cell Biol. Shu S. et. al., (2012), “Immunogenicity of allogeneic mesenchymal stem cells” J. Cell. Mol. Med. Vol 16, No 9, pp. 2094-2103. Shuvalova N. S. et. al., (2013), “Maintenance of mesenchymal stem cells culture due to the cells with reduced attachment rate” Biopolymers and Cell. Vol. 29. N 1. P. 75-78 Sisakhtnezhad S., Alimoradi E., Akrami H., (2017) External factors influencing mesenchymal stem cell fate in vitro, European Journal of Cell Biology, Vol 96, Issue 1, Pages 13-33. Wagner J E. et. al., (2002) “Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: influence of CD34 cell dose and HLA disparity on treatment-related mortality and survival”. Blood, 100:1611-1618.

(47) TABLE-US-00004 TABLE 1 Each row refers to the product(s) marked by “X” delivered before or after 3D culture for AUTOLOGOUS PRODUCTS FROM ONE DONOR research or Whole Umbilical transplantation in an Serum, Plasma or Umbilical Cord Umbilical Cord Cord Tissue HLA-and/or sex Platelet Lysate Blood Cells Tissue Cells Matrix from matched patient from newborn A from newborn A from newborn A newborn A 1 X DELIVERED BEFORE OR 2 X X AFTER 3D CULTURE IN 3 X X X OR WITHOUT HYPOXIA 4 X X X X FOR RESEARCH OR 5 X X X TRANSPLANTATION IN 6 X X AN HLA- AND/OR SEX 7 X X MATCHED PATIENT 8 X X X 9 X 10 X X 11 X X 12 X X X 13 X 14 X 15 X X

(48) TABLE-US-00005 TABLE 2 Each row refers to the product(s) marked by “X” delivered before or Allogeneic Product after 3D culture for Pooled HLA- and/or research or AUTOLOGOUS PRODUCTS FROM ONE DONOR Sex Matching Whole transplantation in an Serum, Plasma or Umbilical Cord Umbilical Cord Umbilical Cord HLA-and/or sex Platelet Lysate Blood Cells Tissue Cells Tissue Matrix matched patient from newborn A from newborn A from newborn A (taken from donors) 1 X DELIVERED BEFORE OR 2 X X AFTER 3D CULTURE IN 3 X X X OR WITHOUT HYPOXIA 4 X X X X FOR RESEARCH OR 5 X X X TRANSPLANTATION IN 6 X X AN HLA- AND/OR SEX 7 X X MATCHED PATIENT 8 X X X 9 X 10 X X 11 X X 12 X X X 13 X 14 X 15 X X

(49) TABLE-US-00006 TABLE 3 Each row refers to the product(s) marked by “X” delivered before or Allogeneic Product after 3D culture for Pooled HLA-and/or AUTOLOGOUS PRODUCTS FROM ONE DONOR research or Sex matching sera/ Whole Umbilical transplantation in an plasmas or platelet Umbilical Cord Umbilical Cord Cord Tissue HLA-and/or sex lysates (taken from Blood Cells Tissue Cells Matrix from matched patient donors) from newborn A from newborn A newborn A 1 X DELIVERED BEFORE OR 2 X X AFTER 3D CULTURE IN 3 X X X OR WITHOUT HYPOXIA 4 X X X x FOR RESEARCH OR 5 X X x TRANSPLANTATION IN 6 X X AN HLA- AND/OR SEX 7 X X MATCHED PATIENT 8 X X X 9 X 10 X X 11 X X 12 X X X 13 X 14 X 15 X X

(50) TABLE-US-00007 TABLE 4 Each row refers to the product(s) marked by Allogeneic Product “X” delivered before or Allogeneic Product Pooled HLA- and/or after 3D culture for Pooled HLA- and/or Autologous Products Sex Matching Whole research or Sex matching sera/ from Same Donor Umbilical Cord transplantation in an plasmas or platelet Umbilical Cord Umbilical Cord Tissue Matrix HLA-and/or sex lysates (taken from Blood Cells Tissue Cells (taken from same matched patient donors) from newborn A from newborn A pool of sera donors) 1 X DELIVERED BEFORE OR 2 X X AFTER 3D CULTURE IN 3 X X X OR WITHOUT HYPOXIA 4 X X X X FOR RESEARCH OR 5 X X X TRANSPLANTATION IN 6 X X AN HLA- AND/OR SEX 7 X X MATCHED PATIENT 8 X X X 9 X 10 X X 11 X X 12 X X X 13 X 14 X 15 X X

(51) TABLE-US-00008 TABLE 5 Each row refers to the product(s) marked by Pooled HLA- and/or “X” delivered before Pooled Umbilical Cord Umbilical Cord Tissue Sex Matching Whole or after 3D culture Pooled HLA- and/or Blood Cells from HLA- Cells HLA- and/or Sex Umbilical Cord Tissue for research or Sex matching sera/ and/or Sex matched matched patient Matrix (taken from same transplantation in an plasmas or platelet patient (taken from (taken from same pool pool of sera and cord HLA-and/or sex lysates (taken from same pool of sera of sera and cord blood blood and cord tissue matched patient donors) donors) donors) cells donors) 1 X DELIVERED BEFORE OR 2 X X AFTER 3D CULTURE IN 3 X X X OR WITHOUT HYPOXIA 4 X X X X FOR RESEARCH OR 5 X X X TRANSPLANTATION IN 6 X X AN HLA- AND/OR SEX 7 X X MATCHED PATIENT 8 X X X 9 X 10 X X 11 X X 12 X X X 13 X 14 X 15 X X