WO2018156746A1 - Methods of processing biological liquid allografts and methods of use - Google Patents

Abstract

The present invention is directed to methods of processing a biological liquid tissue allograft, such as amniotic fluid, and products produced according to these methods. The present invention is also directed to methods of therapeutic use involving such products.

Description

METHODS OF PROCESSING BIOLOGICAL LIQUID ALLOGRAFTS

AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No.

62/462,121, filed on February 22, 2017, the contents of which are incorporated herein by reference in its entirety.

TECHNICAL FIELD

This invention relates to a method for processing biological liquid tissue allografts to optimize the biological payload by concentrating bioactive macromolecules and reducing lower molecular weight contaminants. In particular, the invention applies to tissues such as soluble secreted extracellular matrix present in conditioned cell culture media, amniotic fluid, and potentially other biological fluids. The resultant minimally processed biological liquid allografts can be applied therapeutically in order to provide cushion and pain relief for arthritic joints (for example, as amniotic fluid derived products used in viscosupplementation). Additional applications include supplements for tissue allograft scaffolds that promote engraftment and cellular ingrowth (cell conditioned media). BACKGROUND

Biological tissues can be comprised of specialized layers of living cells, biological fluids, and structural components collectively referred to as the extracellular matrix (ECM). Tissue cells and their progenitors, stem cells, are increasingly used in therapeutic applications for regenerative medicine. The extracellular matrix is a complex milieu that can include specialized structural proteins and embedded growth factors that can act as ligands for cell surface receptors, providing anchorage, adhesion, viscoelasticity or stiffness, cell-to-cell junctions, or a solid substrate for cellular assembly and propagation. Less appreciated perhaps are the biological fluids, which themselves can contain a mixture of cells, soluble components including buffering salts, waste products, ECM proteins, growth factors and cell signaling compounds.

The use of biological fluids can vary widely in their application. Bone marrow lysates, amniotic fluid-derived products, or platelet rich plasma (PRP) is used as injectables for viscosupplementation, anti-inflammation, or tissue regeneration. Such liquid tissue derived products can include cell conditioned media, which begins as a formulation of buffer salts, amino acids, and metabolic precursor macromolecules that is used to bathe and feed cells grown in tissue culture in a laboratory setting. Additional supplementation of this cell media with rich complex compounds such as fetal bovine serum or human platelet lysate is usually required to supply all of the necessary components for maintaining cell viability in vitro. The resultant secreted products and cellular effluent serve to "condition" the surrounding media, which will then also be enriched with metabolites and signaling molecules that are useful in communication with other cells, attracting vasculature via angiogenic growth factors, or specifying differentiation of nearby precursor cells, when transplanted into the body. This conditioned media can also contain soluble secreted factors (notably ECM components) that play a structural as well as a signaling role. However, for viscosupplementation, the American Academy of Orthopedic Surgeons noted in 2013 that they could not recommend using hyaluronic acid for patients with symptomatic osteoarthritis of the knee. This strongly worded guidance was based on a meta-analysis of studies that failed to show clinically significant benefit from traditional HA-based viscosupplementation. Clearly, an alternative that provides longer-term pain relief and benefit to osteoarthritis sufferers is warranted.

Processing of biological fluids to generate products useful in research and therapeutic applications can involve physical and chemical manipulations. In the case of blood, separation of cells, serum, and plasma can be achieved by centrifugation and filtration. Serum itself can be subjected to heat-inactivation, filtration over selectively adherent substrates (carbon-filtration), or purification of entire classes of biomolecules (e.g., immunoglobulin fractions) through column chromatography. The use of physical and chemical fractionation methods to isolate useful components of otherwise complex biological fluids underlies the methodology for generating liquid allografts, bioactive macromolecules, and derivative products.

SUMMARY OF THE INVENTION

The invention is directed to methods of processing biological fluid and compositions produced according to these methods. In some embodiments, the composition further comprises an allograft material, a wound dressing, bandage, or a dermis scaffold. The allograft material may be demineralized bone matrix. In compositions comprising a wound dressing or bandage, the processed biological fluid is applied to the wound dressing or bandage. In the composition comprising a dermis scaffold, the processed biological fluid is infused into the dermis scaffold.

The methods of processing biological fluid comprise providing a biological fluid; separating the biological fluid into a cell pellet portion and a supernatant portion; concentrating the supernatant portion using a tangential cross-flow apparatus to produce a concentrated supernatant, wherein the tangential cross-flow apparatus comprising a cell strainer has a pore size of no more than 100 microns; and diluting the concentrated supernatant to produce a therapeutic concentration of the supernatant. In some implementations, the methods further comprise resuspending the cell pellet portion to produce a cellular component and adding the cellular component to the therapeutic concentration of the supernatant to produce a therapeutic composition. In some aspects, resuspending the cell pellet portion involves adding the supernatant portion to the cell pellet portion.

In some implementation, the methods also comprise adding a cryopreservant to the concentrated supernatant. In some embodiments, the cryopreservant is not DMSO or does not contain any DMSO (DMSO-free). Where the methods comprise adding a cryopreservant, the methods may further comprise cooling the therapeutic concentration of the supernatant or the therapeutic composition to at least -80°C at a rate of no faster than cooling by 1°C per minute. In some implementation, the therapeutic concentration of the supernatant or the therapeutic composition is cooled to -150°C.

In some embodiments, the therapeutic concentration of the supernatant is a 1 : 1 dilution of the concentrated supernatant.

The invention is also directed the methods of using the biological fluid processed according to the described methods. In some implementation, the processed biological fluid is administered to a subject for anti -inflammatory pain relief. In another implementation, the processed biological fluid is administered to a subject for enhanced wound healing or cartilage repair. In such methods, the processed biological fluid may administered topically via application to a wound dressing or bandage that is then applied to the wound on the subject. In another embodiment, the biological fluid may be administered via a dermis scaffold, where the processed biological fluid is infused into the dermis scaffold. In still another implementation, the processed biological fluid is administered to a subject to enhance the regeneration of bone along with an allograft material. The allograft material may be demineralized bone matrix. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a flowchart detailing an overview of an exemplary amniotic fluid processing workflow. The main workflow is depicted with fat solid arrows, while skinnier solid arrows indicate incidental steps. Dotted line arrows denote optional steps representing alternative processes.

Figure 2 depicts the set-up for cross filtration apparatus.

Figure 3 details the results of an experiment demonstrating equivalence in performance between an irradiated and non-irradiated tangential crossflow cassette.

Figure 4 depicts semi-quantitative analysis of the cytokines, chemokines, and other proteins found in full strength (IX) amniotic fluid product produced according to the methods of the invention.

DETAILED DESCRIPTION

As used herein, the verb "comprise" as is used in this description and in the claims and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there is one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one."

As used herein, the term "donor" refers to a human or animal providing biological tissue for transfusion or transplantation, in particular biological fluids.

This invention relates both physical and chemical methods for producing a liquid allograft from a biological fluid (for example, human amniotic fluid, blood plasma, urine, breast milk, or synovial fluid) while assuring preservation of the biological activity of components present in the final processed mixture. The described methods incorporate well-established principles of centrifugation and dialysis as a selective technique to retain and preserve cellular components and soluble bioactive compounds necessary for performance of the final product. The invention also relates to final products produced from such processing of a biological fluid. A. The Methods

The process comprises separating the cellular components of the biological fluid from the liquid components to produce a cell pellet portion and a supernatant portion. This may be done, for example, by gentle centrifugation (for example, less than 350 x g, less than 300 x g, between 50 x g to 400 x g, between 75 x g to 350 x g, between 100 x g to 350 x g, between 100 x g to 300 x g, about 90 x g, about 100 x g, about 120 x g, about 150 x g, about 200 x g, about 300 x g, or about 350 x g) to form a loose pellet. The supernatant portion is then concentrated to a desired level according to use and cell type. The supernatant portion may be concentrated, for example, by a tangential cross-flow apparatus.

In some embodiments, the cell pellet portion is saved for subsequent addition into the concentrated supernatant. Accordingly, in some embodiments, the cell pellet may be stored until it is necessary to add the cellular components back to the liquid components of the biological fluid. In preferred processes, the cellular components are added back after the concentrated supernatant portion is diluted to the desired concentration, for example, via a 1 : 1 dilution. The cell pellet portion may also be further manipulated in order to optimize the final product. This could include washing with biocompatible buffers (such as phosphate buffered saline, PBS) and re-pelleting, for example, via gentle centrifugation. In some aspects, the manipulation includes resuspending the pellet in a special solution for removal of unwanted elements. For example, the pellet may be resuspended in Ammonium Chloride Potassium Red Blood Cell Lysis buffer (155 mM H₄C1, 10 mM KHCO₃, 0.1 mM EDTA) to eliminate contaminating red blood cells. Residual cellular material and particulates in the supernatant can be removed or reduced by filtration through filter of 0.45 μπι pore size into a sterile vessel. In some implementations, this is performed through filtration using a sterile tissue culture membrane bottle top filter. Slightly viscous fluids, like amniotic fluid, may require the membrane composition of the bottle top filter be more permissive towards filtration. For example, the membrane material for filtering slightly viscous fluids like amniotic fluid may be PES or phenylethersulfone membranes, which have asymmetric pores that permit faster flow rates with viscous solutions.

In some implementations, the cell pellet portion is stored in 4°C in a cellular suspension until the processing of supernatant portion of the biological fluid is completed, such as the supernatant portion is concentrated and then diluted to the desired concentration for a specific use in a final product. The cellular suspension is produced by resuspending the cell pellet portion in a small amount of fluid (for example, a 10: 1 ratio of fluid volume to cell pellet volume). Given the cells are already adapted to the osmotic conditions present in the fluid, by retaining them in their native aqueous milieu, the cells are more likely to remain undamaged. Therefore, in some preferred embodiments, the cellular suspension is produced by resuspending the cell pellet portion in the liquid from which they were derived. Alternatively, another biocompatible isotonic buffer could be used, such as phosphate buffered saline (PBS). PBS is preferable as a resuspension solution if a washing step is desired to remove cell debris. Such embodiments of the process are suitable when the final product where the supernatant portion and the cell pellet portion would be combined without any delay in use, optimally no more than 72 hours after recovery from the donor. However, hypothermic stasis or low-temperature cell pausing, when optimized to cell type, can preserve cell viability for 7-10 days.

If there is a delay in use, the cellular components of the biological fluid may be kept in a hypothermic stasis, for example the cell pellet portion is resuspended in a cellular suspension a hypothermic storage solution, for example, HypoThermosol (BioLife Solutions) or ViaSpan (University of Wisconsin solution), and stored at refrigeration temperatures (about 4°C). The composition of these hypothermic storage solutions could include ionic components, such as sodium and chloride salts, pH buffers, energy substrates, and osmotic stabilizers, formulated to balance cellular ion concentrations which are altered upon hypothermia and nutrient deprivation.

In some embodiments, the process includes cryopreservation. Cryopreservation is a controlled rate freezing process. In some implementations, the temperature is depressed at a rate no faster than 1°C per minute, until reaching at least -80°C. Long term storage (a couple of months to years) at -150°C (liquid nitrogen vapor phase) offers even greater assurance of protection against degradation of biological compounds. Cryopreservation occurs through use of a cryopreservant, which inhibits the formation of ice crystals that could damage intact cells. Thus, in some implementations, a cryopreservant may be added to the concentrated supernatant. Cryopreservants are commercially available and are often proprietary mixtures containing dimethyl sulfoxide (DMSO). Although DMSO is established to be a safe and non-stem cell toxic agent, it is associated with negative clinical side effects in some cases. DMSO-free cryopreservants, for example, compositions of carbohydrates (sugars) and sugar alcohol, are also suitable for the processes of the present invention. 1. Amniotic fluid as the biological fluid

In one implementation of the process, amniotic fluid is minimally manipulated to provide a safer, more beneficial clinical product (see Figure 1). As a liquid allograft that can induce an anti-inflammatory state, safely processed amniotic fluid products can be used as injectables to encourage healing. Current modalities for viscosupplementation in arthritic joints use hyaluronic acid, which are believed to restore the cushioning environment of the synovial space. The antiinflammatory properties of stem cells, notably hematopoietic or mesenchymal stem cells present in bone marrow or platelet-rich plasma, have also been used to curb pain and promote regeneration in orthopedic interventions. The intrinsic properties of amniotic fluid address the same needs that existing products already in clinical use do, however, may provide superior benefits in nullifying pain and enhancing patient outcomes. The presence of urea and uric acid in amniotic fluid, however, could confound the effects of the beneficial compounds present. This invention describes unique methodology to selectively remove urea and uric acid, while concentrating the desirable components of amniotic fluid. It also includes ways of retaining viability of cells present in the amniotic fluid, which can also be a complex mixture of different types, but includes cells with stem cell-like properties.

In a preferred embodiment of the present invention, amniotic fluid is recovered from a live donor and processed to: 1) avoid undue damage to the mixture of cells contained therein, 2) concentrate most of the soluble components while removing lower molecular weight waste products accumulated in the gestational environment, 3) provide for subsequent addition of buffering components and cryopreservation of compounds including the cellular components and bioactive macromolecules. The method applied to achieve these goals uses gentle centrifugation to remove cells from the amniotic fluid prior to concentration of the fluid component using tangential crossflow dialysis devices. The resulting concentrated fluid (usually 2X strength) is recombined with cells sequestered during the fluid concentration process and diluted with cryopreservant media to reconstitute a full-strength bioactive whole amniotic fluid that is cryopreserved by subjecting to controlled-rate freezing conditions.

Amniotic fluid is handled in a sterile fashion during the time of birth by recovery agencies skilled in the art. Recovery by Caesarean delivery is recommended as not to incur additional bioburden encountered in the birth canal and vagina. In the case of amniotic fluid, initial processing of the received donor material involves gross filtration to remove meconium and tissue particulates. Using a cell strainer with a pore size of 100 microns allows cells and fluid to be passed and collected, whereas clumps of cells and larger particulate matter is removed. Cells are gently centrifuged to separate them from the fluid component of the amniotic fluid. The cell viability can be maintained by suspending a concentrated cell pellet in a small amount of the fluid component and stored under refrigeration (4°C). Cell viability is stable for nearly 36 hours post-delivery, during which time the fluid component can be processed by tangential crossflow concentration.

If the 36-hour window is untenable, due to shipping times between the site of recovery and the tissue processing facility, cells can be gently centrifuged at the site of recovery, separated from the fluid component, and resuspended in a hypothermic stasis solution. The cell viability window can then be extended to accommodate the delayed delivery to the processing agency, while both the fluid component and the cellular component are preserved under refrigeration or storage on wet ice (4°C). 2. Tangential cross-flow concentration

Processing the fluid component by tangential cross-flow concentration is detailed in Figure 2 and involves specialized dialysis cassettes that are commercially available. The fluid is then pumped through pre-sterilized single use tubing and cassettes. The tangential crossflow concentration process is a design based on specialized organization of dialysis membranes that restrict the passage of higher molecular weight soluble components while letting smaller molecular weight substances through to the effluent. Pore size choice (or the MWCO, molecular weight cutoff) will define the threshold by which components are retained and concentrated or lost. Since the undesirable waste components of amniotic fluid are small, this approach is ideal for concentrating the desirable components in the liquid fraction. Urea and uric acid are roughly -60 and -168 g/mol (molecular weight), respectively. Hyaluronic acid, on the other hand, is a large molecule and its size can range from 10 to 1000 kiloDaltons. This molecular weight differential between beneficial components of amniotic fluid compounds and less desirable ones, makes the application of a tangential crossflow dialysis system an ideal one for enriching those components that can provide benefit.

In another embodiment of the process, a full-strength cryopreserved reconstituted amniotic fluid product is processed. The tangential crossflow concentration process uses tubing, a peristaltic pump, and specialized tangential crossflow dialysis cassettes. To ensure aseptic processing, and as not to introduce any additional bioburden, tubing is autoclaved and designated single-use only. Tangential crossflow cassettes can be irradiated with low-dose gamma radiation to render them sterile, without compromising performance. The results of a proof-of-principle experiment that demonstrates this equivalence in performance is shown in Figure 3.

The aim of employing tangential crossflow dialysis for the preferred embodiment of this invention is to bring the fluid component of the human amniotic fluid to at least a two-fold concentration. This enables equivolume dilution with cryopreservant which restores the fluid component to its original strength. The cellular component of the human amniotic fluid source material can be also added back to reconstitute a "whole" amniotic fluid product. This whole amniotic fluid product is then suitable for cryopreservation and storage at ultracold temperatures prior to clinical use. The preferred range of ultracold temperatures for storage of the whole amniotic fluid products (containing both an amniotic fluid component and intact cells) is -150°C (liquid nitrogen vapor phase). Temperatures to cryopreserve or store the cell-free products could be -150°C (preferred), -80°C (industrial ultralow freezers), or -50°C (shipping on dry ice in insulated containers). As such, in some implementations, ultracold temperature for cryopreservation or storage of cell-free products should be no higher than -50°C. Cell-free compositions of soluble secreted extracellular matrix (conditioned media) could even be stably stored at ranges of -20°C to -80°C.

B. The Final Product

The present invention is also directed to final products produced from biological fluids according to the disclosed processes. In some implementations, the process comprises making a final product, for example a cryopreserved, reconstituted whole human amniotic fluid. The benefit of the final product of the present invention is increasing biocompatibility. Currently, viscosupplements based on hyaluronic acid for use in humans are typically sourced from animal (such as rooster comb HA) or bacterial fermentation sources. These products could contain impurities from those source organisms, whereas the final product of the present application produced with human amniotic fluid would only contain human HA. Thus, it would be more biocompatible to human patients. Additionally, the components of human amniotic fluid are similar in composition to healthy synovial fluid present in the joints. Like synovial fluid, amniotic fluid contains hyaluronic acid in addition to phospholipids, cholesterol, growth factors, cytokines, and stem cells— all of which can contribute to its biological payload and its regenerative properties.

The final product with amniotic fluid may be used in orthopedic applications, including injection into arthritic joints in order to provide anti -inflammatory pain relief and viscosupplementation. By removing low molecular contaminants (e.g. urea and uric acid), the final product should be superior to competitor' s products in which these components are retained. Uric acid is known to be a source of inflammation in occurrences of gout and should be contraindicated for injection into an arthritic joint. The final product can be used at the discretion of a medical professional, doctor, or surgeon to enhance the regeneration of bone by applying it to transplanted allograft material (e.g., demineralized bone matrix products) used in correcting spinal deformities, inducing spinal fusion, and other orthopedic surgeries. The rich growth factor content and regenerative potential of the final product is believed to enhance the rate of fusion, or osteogenesis, when coupled with demineralized bone matrix material.

Human amniotic fluid has been shown to enhance angiogenesis and accelerate wound closure when used to treat difficult-to-heal wounds. Thus, the final product with amniotic fluid (either concentrated or not and either in the presence or absence of the cellular components) may also be for wound healing, for example of diabetic foot ulcers, burns, or skin lesions. The product could be applied topically as part of wound dressing or bandage or infused into a dermis scaffold for soft tissue repair. As a component of human amniotic fluid is hylauronan, which has been shown to promote scarless healing, the final product with amniotic fluid could also be used for cartilage repair. Too often after injury, fibrotic cartilage (scar tissue) is formed instead of functional hyaline cartilage, which has the proper lubricating, load bearing, and structural properties required for joint function.

Another embodiment of this invention provides a multi-dose equivalent in a smaller volume through additional concentration of the fluid component. As shown in Figure 1, which notes the option for further concentration during the tangential crossflow processing of the fluid component. Thus, the endpoint in concentration need not be simply two-fold, but "X-fold" or many folds. The experiment in Figure 3 used 20-fold concentration as an endpoint, which would translate to a ten-fold concentration in a final cryopreserved product (after 1 : 1 dilution with cryopreservant). This allows for a huge increase in biological payload, both by concentrating the soluble growth factors in the fluid component and allowing for add-back of cells diverted early in the process from a much greater volume of starting material than the final volume. This concentrated final product (as compared to the biological fluid) could be useful where inj ection of excessive volume of liquid is contraindicated (e.g., in smaller intra-articular joint spaces in the foot and ankle). Conversely, a more concentrated or highly-folded final product could be diluted with an appropriate physiological buffer to a larger volume, where more irrigation or flow is desired, without diluting the biological activity below full (or original) strength.

In another embodiment of the process, a final product that is free of added-back cells may also be produced. As shown in Figure 1, the step of adding back the cellular components may be omitted. Where the biological fluid is amniotic fluid, the cell-free final product could be used topically or in aesthetic applications to curb inflammation and provide tissue restoration, for example, when coupled with facial microneedling.

The present invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference in their entirety for all purposes.

EXAMPLES

Elements and acts in the example are intended to illustrate the invention for the sake of simplicity and have not necessarily been rendered according to any particular sequence or embodiment. The example is also intended to establish possession of the invention by the Inventors.

A. Example Process: Figure 1

Donated amniotic fluid recovered by Caesarean section is aseptically transported to the tissue processor at 4°C whereupon it is centrifuged gently to pellet the cells. The cell pellet is retained for later add-back to the processed fluid component. Generally, the residual cells will be resuspended in a small amount of cell supernatant, but in the cases in which a delayed use of the cells is warranted, cells can be put into hypothermic stasis for longer durations prior to add-back. The fluid component is further processed using a tangential cross-flow apparatus, which results in 2-fold concentrated fluid, if the final product is to be original strength fluid. The tangential flow process also removes undesirable low molecular weight waste [inverted text]. However, the process also allows for further concentration into X-fold (X>2) concentrated fluid, if a higher potency final product is desired. The processed concentrated fluid is diluted 1 : 1 (with respect to final volumes) with cryopreservant prior to add-back of cells. The reconstituted "whole" product is then subjected to controlled rate freezing resulting in a cryopreserved final product.

B. Tangential Crossflow Concentration: Figure 2

The cassette is commercially available with the following specifications: phenyl ethersulfone (PES) membrane composition with molecular weight cutoff (MWCO) of 3000 Daltons. The irradiated cassette was dosed with gamma radiation at 18.5-25.8 kGy. A mock amniotic fluid was made from commercially available chemicals to mimic the constituent concentrations found in human amniotic fluid. These included bovine serum albumin (BSA) at 4 mg/ml, hyaluronic acid at 2500 ng/ml, uric acid at 1200 μπιοΙεβ/Ε, and urea at 50 mg/dL. Components were dissolved in phosphate buffered saline at physiological pH. Concentration of the mock amniotic solution was followed at five different assay points, lx (starting material), 2x (two-fold concentrated), 5x, lOx, and 20x. These assay points were met by observing volume of the concentrated solution versus the original starting volume as waste/effluent was channeled away. Effluent was also analyzed.

Figure 3 Panel A shows efficiency of uric acid and urea (Waste Products) removed from the mock fluid. Figure 3 Panel B (Protein Analysis) shows total protein expressed as percent retained during the course of concentration. Also shown is the concentration of hyaluronic acid in mg/ml. Panel C (Effluent Analysis) shows comparable results achieved from either the irradiated or the non-irradiated cassette with respect to waste products removal. Total protein was lost to the effluent at 25% or 27% respectively. This could indicate loss of degraded or lower molecular weight forms either BSA, since hyaluronic acid was undetectable (N/D) in the effluent.

Claims

CLAIMS What is claimed is:

1. A method of processing a biological fluid comprising:

obtaining the biological fluid;

separating the biological fluid into a cell pellet portion and a supernatant portion;

concentrating the supernatant portion using a tangential cross-flow apparatus to produce a concentrated supernatant; and

diluting the concentrated supernatant to produce a therapeutic concentration of the supernatant.

2. The method of claim 1, wherein the tangential cross-flow apparatus comprises a cell strainer having a pore size of no more than 100 microns.

3. The method of claim 1 or 2, further comprising resuspending the cell pellet portion to produce a cellular component and adding the cellular component to the therapeutic concentration of the supernatant to produce a therapeutic composition.

4. The method of claim 3, wherein resuspending the cell pellet portion comprising adding an amount of the supernatant portion to the cell pellet portion.

5. The method of claim 4, wherein the supernatant portion added to the cell pellet portion is concentrated supernatant.

6. The method of any one of claims 1-4, further comprising adding a cryopreservant to the concentrated supernatant.

7. The method of claim 5, wherein the cryopreservant is not DMSO or is DMSO-free.

8. The method of claim 6 or 7, further comprising cooling the therapeutic concentration of the supernatant or the therapeutic composition to no higher than -80°C.

9. The method of claim 8, wherein cooling occurs at a rate not faster than 1°C per minute.

10. The method of claim 8, wherein the therapeutic concentration of the supernatant or the therapeutic composition is cooled to -150°C.

11. The method of any one of claims 1-10, wherein the therapeutic concentration of the supernatant is a 1 : 1 dilution of the concentrated supernatant.

12. A composition comprising the biological fluid processed using the method of any one of the preceding claims.

13. The composition of claim 12, further comprising an allograft material.

14. The composition of claim 12, further comprising wound dressing or bandage, wherein the processed biological fluid is applied to the wound dressing or bandage.

15. The composition of claim 12, further comprising a dermis scaffold, wherein the processed biological fluid is fused into the dermis scaffold.

16. A method of providing anti-inflammatory pain relief in a subject in need thereof, the method comprising administering to the subject a composition comprising the biological fluid processed using the method of any one of claims 1-10, wherein the biological fluid comprises amniotic fluid.

17. A method of enhancing the regeneration of bone in a subject, the method comprising administering to the subject a composition comprising the biological fluid processed using the method of any one of the claims 1-11, wherein the biological fluid comprises amniotic fluid.

18. The method of claim 17, further comprising an allograft material. The method of claim 18, wherein the allograft material comprises a demineralized bone matrix product.

A method of enhancing wound healing or cartilage repair in a subject, the method comprising administering to the subject a composition comprising the biological fluid processed using the method of any of the claims 1-11, wherein the biological fluid comprises amniotic fluid.

The method of claim 20, wherein the composition further comprises wound dressing or bandage, wherein the processed biological fluid is applied to the wound dressing or bandage.

The method of claim 20, wherein the composition further comprises a dermis scaffold, wherein the processed biological fluid is infused into the dermis scaffold.