WO2021003254A1

WO2021003254A1 - Cryopreservation medium comprising a tissue extract

Info

Publication number: WO2021003254A1
Application number: PCT/US2020/040469
Authority: WO
Inventors: Rouzbeh R. Taghizadeh
Original assignee: Auxocell Laboratories, Inc.
Priority date: 2019-07-01
Filing date: 2020-07-01
Publication date: 2021-01-07

Abstract

This disclosure provides a cryopreservation medium that includes a tissue extract, and a method of cryopreserving cells and/or tissues using the cryopreservation medium.

Description

CRYOPRESERVATION MEDIUM COMPRISING A TISSUE EXTRACT

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional

Application No. 62/869,466, filed July 1, 2019, the contents of which are hereby incorporated by reference in their entirety.

BACKGROUND

[0002] Cryopreservation is a process in which organelles, cells, tissues, extracellular matrix, organs or any other biological material susceptible to damage caused by unregulated chemical kinetics are preserved by cooling to very low temperatures (typically -80 °C using solid carbon dioxide or -196 °C using liquid nitrogen). At low enough temperatures, any enzymatic or chemical activity which might damage the biological material is effectively stopped. Cryopreservation methods seek to reach low temperatures without causing damage by the formation of ice crystals during freezing. For example, the sharp, jagged crystals can pierce and damage the cellular membrane, thereby reducing post-thaw recovery of intact cells.

[0003] Traditional cryopreservation has relied on coating the material to be frozen with a class of molecules termed cryoprotectants, which reduces ice formation during freezing. However, cryoprotectants can become increasingly toxic as concentration increases. (Best (2015) REJUVENATION RESEARCH 18(5):422-436.)

[0004] While the challenges of cryopreservation have been addressed by, for example, attempting to optimize cooling and warming rates or to optimize time of adding individual cryoprotectants during cooling, these strategies are not sufficient to reduce or eliminate the use of cryoprotectants. {Id) Accordingly, cryoprotectants’ toxicity remains an impediment to cryopreservation.

[0005] Accordingly, there is a need in the art to develop an improved cryoprotectant effective for the preservation of tissues, cells, and/or organs. SUMMARY

[0006] This disclosure provides a cryopreservation medium that includes a tissue extract. In some embodiments, the cryopreservation medium is used for cryopreserving cells, tissues and/or organs. In certain embodiments, the tissue extract is a Wharton’s Jelly matrix extract. In certain embodiments, the tissue extract is a placental extract. In some embodiments, the tissue extract ( e.g ., the Wharton’s Jelly matrix extract) includes at least one component selected from glycosaminoglycans (GAGs), hyaluronic acid, collagen, a growth factor, and a cytokine. In some embodiments, the tissue extract is prepared from a mechanical and/or enzymatic digestion of an umbilical cord tissue. In some embodiments, the tissue extract is prepared from an enzymatic digestion of a tissue. In some embodiments, the tissue extract is prepared from a mechanical digestion of a tissue without enzymatic digestion.

[0007] In some embodiments, the cryopreservation medium includes other components in addition to the tissue extract. For example, the cryopreservation medium can also include a serum (e.g., fetal bovine serum, human serum, and human serum albumin), a cryoprotectant (e.g, DMSO, glycerol, ethylene glycol, polyvinylpyrrolidone, propylene glycol, and 2- methyl-2,4-pentanediol), a dextran, and/or a sugar (e.g, sucrose and glucose).

[0008] In some embodiments, the cryopreservation medium does not comprise a cryoprotectant other than the tissue extract (i.e., the cryoprotectant consists of the tissue extract), which can include at least one of glycosaminoglycans (GAGs), hyaluronic acid, collagen, a growth factor, and a cytokine.

[0009] In another aspect, this disclosure provides a method of cryopreserving a cell, a tissue, wherein the method includes freezing the cell and/or tissue in the cryopreservation medium described herein. In some embodiments, the cell and/or tissue is a mammalian cell and/or tissue, e.g, a human cell and/or tissue. In some embodiments, the cell is isolated from an umbilical cord or Wharton’s Jelly, e.g. , the cell comprises a mesenchymal stem/stromal cell or a Wharton’s Jelly stem cell, or from a placenta.

[0010] In another aspect, this disclosure provides a method of cryopreserving a cell or a tissue, wherein the method includes mechanically and/or enzymatically digesting a tissue, releasing the contents of the tissue to form a tissue extract, and freezing a cell or a second tissue in the tissue extract. In certain embodiments, the method includes mechanically and/or enzymatically digesting an umbilical cord tissue, releasing Wharton’s Jelly matrix extract from the digested umbilical cord tissue, and freezing the cell or a second tissue in the Wharton’s Jelly matrix extract. In certain embodiments, the method includes mechanically and/or enzymatically digesting placental tissue, releasing placenta extract from the digested placental tissue, and freezing the cell or a second tissue in the Wharton’s Jelly matrix extract. In some embodiments, the tissue extract is separated from the digested tissue, for example, by sedimentation and/or filtration. In some embodiments, the tissue extract is not separated from the digested tissue and all of the components of the digestion (the digested tissue, the cell and/or undigested tissue, and the tissue extract) are present in the same container and cryopreserved, e.g, without an additional cryoprotectant. In some embodiments, the cell and/or tissue is a mammalian cell and/or tissue, e.g., a human cell and/or tissue. In some embodiments, the cell is isolated from an umbilical cord or Wharton’s Jelly, e.g. , the cell comprises a mesenchymal stem/stromal cell or Wharton’s Jelly stem cell. In certain embodiments, the cell comprises a population of mesenchymal stem cells and other cells, and the method results in a higher percent viability of the population of mesenchymal stem cell as compared to the total population of cells.

[0011] In certain embodiments, the freezing step comprises slow-rate freezing. In certain embodiments, the freezing step comprises vitrification.

BRIEF DESCRIPTION OF THE FIGURES

[0012] Other features and advantages of the present invention, as well as the invention itself, can be more fully understood from the following description of the various

embodiments, when read together with the accompanying drawings, in which:

[0013] FIGS. 1A-D provide graphs showing post-thaw percent viability (FIG. 1A-B) and percent mesenchymal stem cells (MSCs; FIG. 1C-D) of cells cryopreserved with Wharton’s Jelly matrix extract and/or DMSO. In each graph, the dotted line shows results for cells frozen with DMSO only at the concentrations indicated, and the sold line shows results for cells frozen with Wharton’s Jelly matrix extract and DMSO at the concentrations indicated. The leftmost point of the sold line shows data from cells frozen with Wharton’s Jelly alone (i.e., 0% DMSO). FIGS. IB and D depict results from cells that were thawed and then rinsed with saline, whereas FIGS. 1 A and C depict results from cells that were thawed without a saline wash.

DETAILED DESCRIPTION

[0014] The disclosure provides compositions comprising a cryopreservation medium that includes a tissue extract such as a Wharton’s Jelly matrix extract, and a method of cryopreserving cells, tissues and/or organs using compositions described herein. The cryopreservation medium offers certain advantages, including reduced toxicity and improved post-thaw recovery of the cells, tissues and/or organs. Further, the tissue extract comprises extracellular matrix components (ECM) and other proteins that naturally make up and form the structural, supportive environment where cells reside. For example, Wharton’s Jelly matrix extract comprises glycosaminoglycans (GAGs), hyaluronic acid, collagen and other proteins that naturally make up and form the structural, supportive environment where Wharton’s Jelly cells reside in utero. Without wishing to be bound by the theory, inclusion of a tissue extract, e.g ., Wharton’s Jelly matrix extract, in a cryopreservation medium

advantageously provides support and nutrients which can improve post-thaw recovery of cells. Further, inclusion of a Wharton’s Jelly matrix extract in a cryopreservation medium may reduce damage to cells that occurs during freezing at least in part because the

hydrophilic hyaluronic acid in Wharton’s Jelly matrix extract draws in water to reduce crystallization during freezing and, thus, acts as a cryoprotectant.

[0015] To provide an overall understanding of the invention, certain illustrative embodiments will now be described. However, it will be understood by one of ordinary skill in the art that the compositions and methods described herein may be adapted and modified as appropriate and may be employed in other suitable applications. All such adaptations and modifications are to be considered within the scope of the invention.

[0016] A tissue extract is a product derived from digestion of a tissue by any method that digests tissue and separates the tissue into cells, undigested tissue and tissue extract that is essentially or completely depleted of cells. Accordingly, in certain embodiments, the tissue extract is separated from the digested tissue, for example, by sedimentation and/or filtration. However, in other embodiments, the tissue extract is not separated from the digested tissue and all of the components of the digestion (the digested tissue, the cell and/or undigested tissue, and the tissue extract) are present in the same container and cryopreserved, e.g. , without an additional cryoprotectant. The tissue can be isolated from a mammal, for example, a human, a non-human primate, a dog, a cat, a cow, a horse, and a pig.

[0017] In certain embodiments, the tissue extract is a Wharton’s Jelly matrix extract, which is a product derived from an umbilical cord tissue by any method that digests the umbilical cord tissue and separates the umbilical cord tissue into cells, undigested umbilical cord tissue and Wharton’s Jelly matrix extract that is essentially or completely depleted of cells. Accordingly, in certain embodiments, the Wharton’s Jelly matrix extract is separated from the digested umbilical cord tissue, for example, by sedimentation and/or filtration. However, in other embodiments, the Wharton’s Jelly matrix extract is not separated from the digested umbilical cord tissue and all of the components of the digestion (the digested umbilical cord tissue, the cell and/or undigested umbilical cord tissue, and the Wharton’s Jelly matrix extract) are present in the same container and cryopreserved, e.g. , without an additional cryoprotectant. The umbilical cord tissue can be isolated from a placental mammal, for example, a human, a non-human primate, a dog, a cat, a cow, a horse, and a pig.

Tissue Matrix Extract

[0018] Tissue extract can be derived from an umbilical cord tissue using any method known in the art. For example, tissue extract can be derived by enzymatic digestion of a tissue (e.g., an umbilical cord tissue in the case of Wharton’s Jelly matrix extract or placenta in the case of placental extract), such as by a collagenase, a hyaluronidase, a dispase and/or one or more other proteases. In addition, tissue digestion may be facilitated by acids.

Optionally, before the enzymatic digestion, the tissue can be dissected to remove any arteries and veins, and then processed to maximize the available surface area. This processing can generally involve any manner of mechanically increasing the surface area of the tissue, but most often involves finely cutting or mincing the tissue into small strands or pieces, such as with dissecting scissors or a scalpel. Mechanically processing the tissue increases the surface area for tissue digestion and, therefore, reduces the incubation times with enzyme(s). Longer incubation time with the enzyme(s) can obviate the need to initially mechanically process the tissue, given enough enzyme is added. After the digestion, a neutralizing agent, e.g, a serum or bovine albumin, can optionally be added to the digested mixture to neutralize the enzyme(s),

[0019] Another method involves mechanical digestion of a tissue (e.g, an umbilical cord tissue or placental tissue), resulting in a mixture of cells, tissue and tissue extract (e.g, Wharton’s Jelly matrix extract or placental extract). The mechanical digestion can be used alone or in combination with the enzymatic digestion described above. The mechanical digestion can be achieved by any means known in the art, including, e.g, finely cutting and/or mincing the tissue into small strands or pieces, such as with dissecting scissors or a scalpel. Optionally, before the mechanical digestion, a tissue (e.g, an umbilical cord tissue) can be dissected to remove the arteries and veins.

[0020] Prior to or during digestion, an antibiotic or an antiseptic can be added to the tissue. Antibiotics can include gentamicin, penicillin, streptomycin, amphotericin B, and ciprofloxacin. In certain embodiments, the antibiotic is present in an amount between about 0.1 mg/mL and about 100 mg/mL or about 0.1 IU/mL and about 100 IU/mL. In certain embodiments, an antiseptic such as chi orhexi dine ( e.g ., 0.04% chi orhexi dine) is added to the tissue prior to or during digestion.

[0021] After an enzymatic and/or mechanical digestion, any remaining fragments of undigested tissue can be removed. Depending on their size, undigested tissue can be removed by physical extraction (e.g., with forceps), decanting, aspiration, sedimentation (optionally accelerated by centrifugation), and/or filtering, for example. The cells from the digested tissue can optionally be separated from the homogeneous mixture containing the digested tissue, for example, by sedimentation. Although gravity sedimentation can be used, the sedimentation process can be accelerated by, for example, a centrifuge to enhance the downward movement of the cells through (and, in some sense, out of) the mixture. Upon removal of the cells, the remaining solution is the tissue extract (e.g, Wharton’s Jelly matrix extract or placental extract). Because the digested tissue is generally viscous, it can be washed or diluted with an appropriate sterile solution (such as a buffered salt solution) at any stage in the process.

[0022] Tissue extract can also be derived from an tissue using a tissue processing system, e.g., the AC:Px System (see Taghizadeh et al., CELL TRANSPLANTATION 181-193, 2018). The AC:Px System is a sterile, closed, single-use, completely disposable system to process solid tissue without the need for any enzymatic digestion. The AC:Px System consists of a closed mincer (i.e., semi-automated scissors) and a series of closed bags. It allows standardized processing of a tissue without the use of enzymes in a closed system, and results in fractionation of cells, minced tissue and tissue extract.

[0023] The tissue extract can contain extracellular matrix components (ECM) and other proteins that naturally make up and form the structural, supportive environment where cells reside. For example, Wharton’s Jelly matrix extract can contain glycosaminoglycans (GAGs); hyaluronic acid; collagen; one or more growth factors, e.g, insulin-like growth factor I (IGF-I), fibroblast growth factor (FGF), basic FGF, transforming growth factor beta (TGF-beta), platelet-derived growth factor (PDGF) and epidermal growth factor (EGF); and/or one or more cytokines present in the umbilical cord tissue.

Cryopreservation Medium

[0024] The disclosure further relates to a cryopreservation medium that includes the tissue extract described herein. In some embodiments, the cryopreservation medium includes the tissue extract described herein, optionally another cryoprotectant, and one or more additional components, such as a serum, a dextran, and a sugar. In certain embodiments, the tissue extract is the only cryoprotectant included in the cryopreservation medium. In other embodiments, additional cryoprotectants are included in the cryopreservation medium. When additional cryoprotectants are used, they can in some instances be added in smaller amounts than would be required in the absence of tissue extract.

[0025] In certain embodiments, components of the cryopreservation medium are added separately to cells rather than mixed together prior to addition to cells. Unless otherwise noted, percentages specified throughout the text are percentages of the final cryopreservation medium into which cells are suspended, regardless of whether the components of the cryopreservation medium were added separately to cells or mixed together prior to addition to cells.

[0026] In certain embodiments, tissue extract is included in the cryopreservation medium in an amount between about 1% and about 100%, between about 10% and about 100%, between about 50% and about 100%, between about 90% and about 100%, between about 95% and about 100%, between about 97.5% and about 100%, or about 100%. In certain embodiments, the tissue extract is included in the cryopreservation medium in an amount between about 1% and about 90%, between about 10% and about 90%, between about 50% and about 90%, between about 30% and about 50%, between about 1% and about 50%, between about 5% and about 40%, between about 10% and about 30%, or between about 15% and about 25%. Unless indicated otherwise, percentages are expressed as (v/v).

[0027] In certain embodiments, the cryopreservation medium includes or optionally includes an additional cryoprotectant, for example, DMSO, glycerol, ethylene glycol, polyvinylpyrrolidone, propylene glycol, and 2-methyl-2,4-pentanediol. In some

embodiments, the additional cryoprotectant is selected from DMSO, glycerol, ethylene glycol, polyvinylpyrrolidone, propylene glycol, and 2-methyl-2,4-pentanediol. In some embodiments, the additional cryoprotectant is a combination of at least two cryoprotectants selected from DMSO, glycerol, ethylene glycol, polyvinylpyrrolidone, propylene glycol, and 2-methyl-2,4-pentanediol. The final concentration of additional cryoprotectant in the cryopreservation medium can be, for example, between up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 5%, up to about 2.5%, or up to about 1%. In certain embodiments, the final concentration of additional cryoprotectant in the cryopreservation medium is between about 1% and about 90%, between about 10% and about 80%, between about 20% and about 70%, between about 30% and about 50%, between about 1% and about 50%, between about 5% and about 40%, between about 10% and about 30%, between about 15% and about 25%, between about 5% and 10%, between about 2.5% and 10%, between about 2.5% and 5%, between about 1% and about 10%, between 1% and about 5%, or between about 1% and about 2.5%. In certain embodiments, the final concentration of additional cryoprotectant in the cryopreservation medium is between about 0.01% and about 2.5% or between about 0.01% and about 5%. In certain embodiments, the final concentration of additional cryoprotectant in the cryopreservation medium is about 2.5% or 5%. In certain embodiments, a cryopreservation medium may include tissue extract and about 2.5%, 5% or 10% DMSO. In certain embodiments, a cryopreservation medium may include tissue matrix extract and about 2.5%, 5%, or 10% glycerol. In certain embodiments, the cryopreservation medium comprises no additional cryoprotectant.

[0028] Serums suitable for use herein include any serum used in cryopreservation, for example, a fetal bovine serum, a human serum, a human serum albumin, a horse serum, and a goat serum. In some embodiments, the serum is selected from a fetal bovine serum, a human serum, a human serum albumin, a horse serum, and a goat serum. In some embodiments, the serum is a combination of at least two serums selected from a fetal bovine serum, a human serum, a human serum albumin, a horse serum, and a goat serum. The final serum

concentration in the cryopreservation medium can be between about 1% and about 90%, between about 10% and about 90%, between about 50% and about 90%, or between about 70% and about 50%.

[0029] Dextrans suitable for use herein include any dextran used in cryopreservation, for example, Dextran 10, Dextran 40, Dextran 70, and Dextran 500. In some embodiments, the dextran is selected from Dextran 10, Dextran 40, Dextran 70, and Dextran 500. In some embodiments, the dextran is a combination of at least two dextrans selected from Dextran 10, Dextran 40, Dextran 70, and Dextran 500. The final dextran concentration can be, for example, between about 1% and about 50%, between about 1% and about 40%, between about 1% and about 30%, between about 1% and about 20% or between about 1% and about 10%. For example, a cryopreservation medium can include tissue extract and about 1%, about 2%, about 5%, or about 10% Dextran 40.

[0030] Sugars suitable for use herein include, for example, a sucrose, glucose, fructose, lactose and raffinose. In some embodiments, the sugar is selected from sucrose, glucose, fructose, lactose and raffinose. In some embodiments, the sugar comprises a combination of at least two sugars selected from sucrose, glucose, fructose, lactose and raffinose. The final sugar concentration in the cryopreservation medium can be between about 10 mM and about 500 mM, between about 50 mM and about 400 mM, between about 100 mM and about 300 mM, or between about 150 mM and about 250 mM. For example, a cryopreservation medium may include tissue extract and about 200 mM sucrose. Alternatively, a

cryopreservation medium may include tissue extract and about 200 mM sucrose.

[0031] In some embodiments, the cryopreservation medium described herein further includes one or more of commercially available cryopreservation media, e.g., CTS™ Synth- a-Freeze™ Medium, Recovery™ Cell Culture Freezing Medium, Synth-a-Freeze™

Cryopreservation Medium (Thermo Fisher Scientific), CryoStor® cell cryopreservation media (Sigma-Aldrich), BloodStor® Freezing Media, HypoThermosol® FRS Preservation Media, mFreSR™ and FreSRS™ Cryopreservation Media, MesenCult™-ACF

cryopreservation media, STEMdiff™ Neural Progenitor Freezing Medium (STEMCELL Technologies).

[0032] In certain embodiments, the cryopreservation medium further includes an antibiotic or an antiseptic. For example, the cryopreservation medium can include an antibiotic including gentamicin, penicillin, streptomycin, amphotericin B, and ciprofloxacin. In certain embodiments, the antibiotic is present in an amount between about 0.1 mg/mL and about 100 mg/mL or about 0.1 IU/mL and about 100 IU/mL. In certain embodiments, the cryopreservation medium includes an antiseptic such as chi orhexi dine (e.g, 0.04%

chlorhexidine).

Methods of Cryopreservation

[0033] In certain embodiments, a cryopreservation medium including a tissue extract (e.g, a Wharton’s Jelly matrix extract or a placental extract) in lieu of a traditional cryoprotectant such as DMSO, can be used in a cryopreservation method. In certain embodiments, a cryopreservation medium including a Wharton’s Jelly matrix extract is used in a cryopreservation method. In certain embodiments, a cryopreservation medium including a placental extract is used in a cryopreservation method. In certain embodiments, a cryopreservation medium including a Wharton’s Jelly matrix extract and a placental extract is used in a cryopreservation method. Such methods may result in cryopreservation and cryoprotection of cells (e.g, the native cellular population of UCT) at levels higher than traditional cryopreservation (e.g, DMSO) alone (without tissue extract).

[0034] Accordingly, in another aspect, the application provides a method of

cry opreserving a cell, a tissue or an organ, wherein the method includes freezing the cell, tissue, or organ in the cryopreservation medium described herein. In certain embodiments, an umbilical cord or a portion thereof is mechanically and/or enzymatically digested, releasing the tissue extract from the tissue and forming a mixture of digested tissue, cells, and tissue extract. The cells may be present in the tissue and/or the mixture may contain cells that have been released from the tissue during the digestion process. The mixture may be present in a single container. In certain embodiments, the mixture is cryopreserved without further addition of cryoprotectants (i.e., the cryopreservation medium consists of tissue extract). In certain embodiments, one or more cryoprotectants (in addition to the tissue extract), a dextran, and/or a sugar is added and the mixture is cryopreserved. In this embodiment, the cryoprotectant, the dextran, and/or the sugar may be added in smaller amounts than would be used in the absence of tissue extract.

[0035] In other embodiments, tissue extract is isolated together with one or more of the components of the tissue following mechanical and/or enzymatic digestion. For example, tissue extract and released cells may be isolated from digested (and/or undigested) tissue, and the mixture cryopreserved without further addition of cryoprotectants. In certain

embodiments, one or more cryoprotectants (in addition to the tissue extract), a dextran, and/or a sugar is added and the mixture is cryopreserved. In this embodiment, cryoprotectant, a dextran, and/or a sugar may be added in smaller amounts than would be used in the absence of tissue extract.

[0036] In certain embodiments, tissue extract is substantially purified from mechanically and/or enzymatically digested tissue and is essentially cell-free. The tissue extract may be combined with any cell or tissue, optionally in combination with another cryoprotectant and/or a dextran, and/or a sugar, and cryopreserved.

[0037] The method of cryopreserving a cell, a tissue or an organ described herein may include one or more of the following processes in order to obtain high survival on thawing of the cell, tissue or organ. For example, controlled-rate or slow-rate cooling can be used to allow biological samples to equilibrate to optimal physical parameters osmotically in the cryopreservation medium before cooling in a predetermined, controlled way. Lethal intracellular freezing can be avoided if cooling is slow enough to permit sufficient water to leave the cell during progressive freezing of the extracellular fluid. The cooling rate differs between cells of differing size and water permeability: a typical cooling rate around 1 °C/minute is appropriate for many mammalian cells after treatment with a cryoprotectant, but the rate is not a universal optimum and can be optimized for each type of cell, tissue and organ. For example, the slowing cooling can be performed by placing the final product at 4°C for 30 minutes, then subsequently transferring to -20°C for an additional 60 minutes, then subsequently transferring to -80°C for at least 60 minutes, and then finally subsequently transferring to -196°C for long-term storage. A cool-rate freezer (CRF) can also be utilized to control the rate of freezing. Devices such as a Mr. Frosty™ or CoolCell® can be used to slowly cool the cells at a l°C/min rate.

[0038] In certain embodiments, vitrification can be used. (See, e.g ., Heo et al. (2015) TECHNOLOGY (SINGAP WORLD SCI) 3(1): 64-71; Zhang et al. (2011) NANOMEDICINE (LOND). 6(6): 1115-1129.) Briefly, use of vitrification prevents formation of ice crystals and the increase in ionic strength of unfrozen concentrated solutions. In vitrification, a solution of cells is converted from the liquid phase directly into a glass-like solid. Current vitrification techniques require higher cryoprotectant concentrations (e.g, 6-8 M), but inclusion of Wharton’s Jelly matrix extract can lessen the amount of traditional cryoprotectant (e.g, an additional cryoprotectant) required. Vitrification also requires higher cooling rates (e.g, -1500°C/min) compared with slow freezing methods, which can be achieved by immersing cells/tissues directly into liquid nitrogen (-196°C) or liquid nitrogen vapor (-160°C).

[0039] Cryopreserved samples are stored at temperatures equal to or below -20 °C, for example, -20 °C, between -20 °C to -80 °C, between -80 °C to -136 °C, or between -136 °C and -196 °C. While temperatures between -20 °C to -136 °C are used for cryopreservation, generally temperatures between -136 °C and -196 °C are required for long-term preservation of the more complex biological structures to virtually stop all biological activity.

[0040] In an exemplary cryopreservation method, the cryopreservation medium (e.g, 90% Wharton’s Jelly matrix extract and 10% DMSO concentration (v/v), 50% Wharton’s Jelly matrix extract, 39% serum, 10% DMSO and 1% Dextran-40 (v/v), or 100% Wharton’s Jelly matrix extract, 0% DMSO) is placed at room temperature or chilled on ice, and the cryopreservation medium is added to cells/tissues drop-wise to the appropriate final cryopreservation volume, depending on the type of cryopreservation vehicle used. For instance, cells/tissues can be cryopreserved in one or more cryovials/ampules at various volumes from 1 ml and higher (e.g, 1.5 ml, 2 ml, 2.5 ml, 5 ml, 10 ml, 25 ml, 100 ml or ranges between any of the foregoing values) or in one or more cryobags (e.g, segmented cryobags) that have a volume capacity of 1 mL and higher (e.g, 1 mL - 100 mL, 25 mL-100 mL, etc.). The cryopreservation medium mixture is carefully mixed and transferred into its final cryopreservation ampule or bag (if being prepared outside of the final cryopreservation tube/bag). The final cryopreservation ampule/bag is slowly cryopreserved, typically at a rate of 1 °C/min. The final cryopreserved product is placed at -196 °C for long-term storage and/or until needed for future use.

[0041] Any biological materials, including cells, tissues and organs, can be cryopreserved using the cryopreservation medium described herein. In some embodiments, the cells, tissues and organs are mammalian, e.g., human or non-human, such as murine, simian, equine, bovine, porcine, canine, feline. In some embodiments, the cells are isolated from adipose tissue, kidney, liver, pancreas, placenta, heart, brain, skin, lung, muscle, cartilage, or bone. In some embodiments, the cells are isolated from umbilical cord tissue, Wharton’s Jelly, and/or umbilical cord blood, e.g, umbilical cord blood cells, umbilical cord matrix cells, umbilical cord-derived stem cells including mesenchymal stem cells, and Wharton’s Jelly stem cells. The biological materials may also comprise embryos at cleavage stage (that are 2, 4 or 8 cells) or at blastocyst stage, oocytes such as human oocytes, various stem cells; semen, testicular tissues, ovarian tissues, or placental cells or tissue. In certain embodiments, the cells comprise stem cells, (e.g, embryonic and/or adult stem cells), red blood cells (e.g, erythrocytes), white blood cells (e.g, granulocytes such as neutrophils, eosinophils, basophils and agranulocyes (such as monocytes and lymphocytes), platelets (e.g, fragments of megakaryocytes), nerve cells (e.g, neurons and neuroglial cells), muscle cells (e.g, skeletal, cardiac and smooth), cartilage cells (e.g, chondrocytes), bone cells (e.g, osteoblasts, osteoclasts, osteocytes, and lining cells), skin cells (e.g, keratinocytes, melanocytes, Merkel cells, and Langerhans cells), endothelial cells (e.g, cells lining the blood vessels), epithelial cells (e.g, cells lining body cavities), pericytes, fat cells (e.g, white adipocytes and brown adipocytes), and sex cells (e.g, spermatozoa and ova).

[0042] In certain embodiments, inclusion of a tissue extract improves the recovery of post-cryopreserved cells and/or tissues. In certain embodiments, inclusion of a tissue extract increases the viability of post-cryopreserved mesenchymal stem cells. In certain

embodiments, inclusion of a tissue extract increases the viability of post-cryopreserved mesenchymal stem cells by about 5% to about 20%, 5% to about 15%, 10% to about 20% or 10% to about 15% compared to equivalent post-cryopreserved mesenchymal stem cells not containing the tissue extract. In certain embodiments, the addition of a tissue extract (e.g, a Wharton’s Jelly matrix extract) results in a higher percent viability of the total cell population and/or percent mesenchymal stem cells present in the post-cryopreserved cellular product as compared to an equivalent post-cryopreserved cellular product not containing the tissue extract. EXAMPLES

[0043] The invention now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and is not intended to limit the invention.

[0044] Umbilical cord tissue processed using the mechanical AC:Px® System

fractionates human tissue into its various components, including the native cells that reside within the tissue, as well as the extracellular matrix (ECM) proteins that provide support, growth factors, cytokines, various glycosaminoglycans (GAGs), and other signaling proteins to the native cells. The examples that follow show that Wharton’s Jelly matrix extract (including the ECM proteins found therein) can provide cryoprotection and cryopreservation to the native cells during cryostorage. This protection can be in addition to or in lieu of traditional cellular cryoprotectants (e.g, dimethyl sulfoxide; DMSO).

Tissue Collection and Preparation for Wharton’s Jelly Matrix Extract Production

[0045] Umbilical cord tissues (UCTs) were obtained from healthy mothers who donated their de-identified UCTs after giving written informed consent. UCT was collected after term delivery. Once collected, the UCT (unit) was placed in a sterile specimen jar and delivered to the laboratory for processing. Upon arrival at the processing laboratory, 10 mL of antibiotics (25 mg/mL gentamicin, 100 IU/mL penicillin, 100 mg/mL streptomycin, 0.25 mg/mL amphotericin B, 10 mg/mL ciprofloxacin) was added to the unit and placed at 4 °C until processed— which never exceeded 72 h post-delivery. Immediately prior to processing, the specimen jar containing the unit was taken out of 4°C, sterilized using 70% ethanol and placed in the biosafety cabinet. Chlorhexidine (0.04%) was added for 10 minutes

immediately prior to processing. The unit was carefully taken out of the specimen jar and placed on a sterile petri dish using sterile stainless steel forceps. Using a sterile pair of umbilical cord scissors, the unit was cut into equal quarter segments. Every other quarter was placed into one of the 2 tared tubes— in order to control intra-tissue variability. Tubes were randomly designated for either enzymatic digestion or processing using the AC:Px System. Wharton’s Jelly Matrix Extract Produced by Enzymatic Digestion

[0046] Umbilical cords designated for enzymatic digestion were minced using umbilical cord scissors in their entirety until the tissue was finely minced. The minced tissue was placed into a tared 50 mL conical tube for weight determination post-mincing. A 10 mL solution of 2.5 mg/mL collagenase NB6 in 2 mM calcium chloride (Amresco®, VWR Life Science, Dublin, Ireland) in Dulbecco’s phosphate-buffered saline (DPBS) was added to the 50 mL conical tube containing the minced UCT. The minced tissue was placed in a 37 °C incubator with a rocking platform for 3 hours. In post-incubation, the unit was diluted with 30 mL DPBS and filtered through a 100 micron Steriflip® (Millipore Sigma, Burlington, MA). Undigested minced tissue that did not pass through the filter was collected. After the initial pass through the filter, 50 mL DPBS was added back to the tube containing the digested minced tissue for subsequent washes and refiltered through the same Steriflip® filter for a total of 3 washes. Undigested minced tissue that did not pass through the filter was pooled and collected for cryopreservation. The filtered cell product was placed in a 225 mL conical tube and centrifuged for 20 minutes at 750 g in a centrifuge. The supernatant ( i.e ., Wharton’s Jelly matrix extract) was collected into several 50 mL conical tubes. The cell pellet was collected for cryopreservation.

Wharton’s Jelly Matrix Extract Produced by Mechanical Digestion Using the AC:Px System

[0047] UCTs designated for non enzymatic processing were placed in the AC:Px

(AuxoCell, Cambridge, MA, USA) System. Briefly, the entire tissue was placed in the input chamber of the AC:Px Mincer with the output chamber filled with 0.9% sodium chloride saline. After subsequent mincing and washes with saline, the postminced UCT was transferred into the supplied series of AC:Px bag sets in order to filter and centrifuge the native cellular product. Filtration took place in the AC:Px filter bag that filters using a 100 micron mesh, and subsequent centrifugation took place in the AC:Px centrifuge bag, clipped on a 97 mm blood bag centrifuge adaptor (Beckman Coulter) suspended, using the AC:Px centrifuge clip (AuxoCell). The cells were centrifuged for 20 mins at 750 g in an Allegra X15R (Beckman Coulter) benchtop centrifuge. After centrifugation, the supernatant {i.e., decellularized Wharton’s Jelly matrix extract) was decanted into the AC:Px filter bag, and the cell pellet was collected for cryopreservation. The minced tissue was collected from the AC:Px for cryopreservation. Cryopreservation Methods

[0048] Cells and undigested tissues were isolated from UCT as described above. Cells from umbilical cord tissue (UCT) were cryopreserved in 1 mL Wharton’s Jelly matrix extract (including ECM proteins within the UCT) alone or together with 0%-30% DMSO. The cryopreservation medium was chilled on ice, and the cold cryopreservation medium was added to cells/tissues drop-wise to the final cryopreservation volume. Cells were

cryopreserved in cryovials/ampules. The final cryopreservation ampule was slowly cryopreserved by placing the cells at 4 °C for 20 minutes, then immediately to -20 °C for 90 minutes, then -80 °C for 60 minutes. The final cryopreserved samples were placed at -196 °C and then tested for post-thaw viability as described below.

Comparison of Rates of Post-Cry opreservati on Cell Viability using Wharton’s Jelly Matrix Extract and/or Standard Cryoprotectants DMSO)

[0049] As described above, native cells from umbilical cord tissue (UCT) were cryopreserved with Wharton’s Jelly matrix extract (including the ECM proteins therein) in lieu of DMSO and at varying DMSO concentrations, from a 0%-30% DMSO concentration range. After 48 hours, the cells were thawed with or without a post-thaw wash with 0.9% sodium chloride (NaCl) saline. Cells were analyzed for viability using the viability dye 7- aminoactinomycin-D (7-AAD) to determine how well the Wharton’s Jelly matrix extract protected the cells from damage caused by freezing and thawing. In addition, the percentage of mesenchymal stem cells (MSCs) present in present in the post-cryopreserved cellular product was determined using Anti-CD45, Anti-CD34, Anti-HLA-DR, Anti-CD 1 lb, Anti- CD^, Anti-CD29, Anti-CD105, Anti-CD73, and Anti-CD90 fluorescence conjugated antibodies (all from BD Biosciences) were used to stain cells. Cells were analyzed on an Attune (Therm oFisher, Waltham, MA, USA) flow cytometer.

[0050] The addition of Wharton’s Jelly matrix extract as a cryoprotectant to the native cells with no addition of DMSO (0% DMSO) overall resulted in both relatively higher percent viability of the total cell population and percent MSCs present in the post- cryopreserved cellular product. This was observed with (FIG. IB, D) and without (FIG. 1A, C) a saline wash post-thaw.

[0051] These data suggest that Wharton’s Jelly matrix extract, can be used as a cryoprotectant in lieu of (or in addition to) a traditional cryoprotectant such as DMSO, and that inclusion of Wharton’s Jelly matrix extract in a cryopreservation medium results in cryopreservation and cryoprotection of cells (e.g, the native cellular population of UCT) at levels higher than traditional cryopreservation (e.g, DMSO) alone (without Wharton’s Jelly matrix extract).

INCORPORATION BY REFERENCE

[0052] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

[0053] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMS:

1. A cryopreservation medium comprising a tissue extract.

2. The cryopreservation medium of claim 1, wherein the tissue extract is a Wharton’s Jelly matrix extract.

3. The cryopreservation medium of claim 1, wherein the tissue extract is a placenta extract.

4. The cryopreservation medium of any preceding claim, wherein the Wharton’s Jelly matrix extract comprises at least one of glycosaminoglycans (GAGs), hyaluronic acid, collagen, a growth factor, and a cytokine.

5. The cryopreservation medium of any preceding claim, wherein the tissue extract is prepared from a mechanical digestion and/or enzymatic digestion of tissue.

6. The cryopreservation medium of claim 5, wherein the tissue extract is prepared from a mechanical digestion of the placental tissue or umbilical cord tissue, without enzymatic digestion.

7. The cryopreservation medium of any preceding claim, further comprising a serum.

8. The cryopreservation medium of claim 7, wherein the serum is selected from fetal bovine serum, human serum albumin, and human serum.

9. The cryopreservation medium of any preceding claim, further comprising a

cryoprotectant.

10. The cryopreservation medium of claim 9, wherein the cryoprotectant is selected from DMSO, glycerol, ethylene glycol, polyvinylpyrrolidone, propylene glycol, and 2-methyl-2,4- pentanediol (MPD).

11. The cryopreservation medium of any one of claims 1-8, wherein the cryopreservation medium does not comprise a cryoprotectant other than the tissue extract.

12. The cryopreservation medium of any preceding claim, further comprising a dextran.

13. The cryopreservation medium of any preceding claim, further comprising a sugar.

14. The cryopreservation medium of claim 13, wherein the sugar is sucrose or glucose.

15. A cryopreservation medium comprising a cryoprotectant, wherein the cryoprotectant consists of tissue extract.

16. The cryopreservation medium of claim 15, wherein the tissue extract is a Wharton’s Jelly matrix extract.

17. The cryopreservation medium of claim 15, wherein the tissue extract is a placenta extract.

18. The cryopreservation medium of claim 15, wherein the tissue extract comprises at least one of glycosaminoglycans (GAGs), hyaluronic acid, collagen, a growth factor, and a cytokine.

19. The cryopreservation medium of any one of claims 15-18, wherein the tissue extract is prepared from a mechanical digestion and/or enzymatic digestion of a tissue.

20. The cryopreservation medium of claim 19, wherein the tissue extract is prepared from a mechanical digestion of the tissue, without enzymatic digestion.

21. A method of cryopreserving a cell or a tissue, the method comprising freezing the cell or the tissue in the cryopreservation medium of any one of claims 1-16.

22. A method of cryopreserving a cell or a tissue, the method comprising mechanically and/or enzymatically digesting a tissue, releasing the contents of the tissue to form a tissue extract, and freezing the cell or the tissue in the tissue extract.

23. The method of claim 22, wherein the digested tissue, the cell or tissue, and the tissue extract are present in the same container.

24. The method of claim 22 or 23, wherein the tissue is an umbilical cord and the tissue extract is a Wharton’s Jelly matrix extract.

25. The method of claim 22 or 23, wherein the tissue is a placenta, and the tissue extract is a placenta extract.

26. The method of any one of claims 22-25, wherein the method is performed without a sedimenting step.

27. The method of any one of claims 22-25, wherein the method is performed with a sedimenting step.

28. The method of any one of claims 22-27, wherein the method is performed with a filtering step.

29. The method of any one of claims 21-28, wherein the cell is a mammalian cell.

30. The method of any one of claims 21-29, wherein the cell or the tissue is isolated from an umbilical cord.

31. The method of claim 30, wherein the cell is a Wharton’s Jelly cell.

32. The method of any one of claims 21-31, wherein the cell comprises a mesenchymal stem cell.

33. The method of claim 32, wherein the cell comprises a population of mesenchymal stem cells and other cells, and the method results in a higher percent viability of the population of mesenchymal stem cell as compared to the total population of cells.

34. The method of any one of claims 22-33, wherein the freezing step comprises slow-rate freezing.

35. The method of any one of claims 22-33, wherein the freezing step comprises vitrification.