Methods for processing decellularized amniotic fluid

Abstract

A processing technique for amniotic fluid (AF) described herein is designed to maximize the retention of bioactive components while ensuring efficient decellularization and long-term preservation. The method integrates ultrafiltration, osmotic disruption, and cryopreservation in a streamlined workflow. Key steps involve the sequential use of semi-permeable membranes to enhance filtration efficiency, along with precise centrifugation to ensure thorough cell removal.

Description

METHODS FOR PROCESSING DECELLULARIZED AMNIOTIC FLUIDCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application No.63 / 743,448 entitled ‘METHODS FOR PROCESSING DECELLULARIZED AMNIOTIC FLUID,” filed on January 9, 2025, the contents of which are hereby incorporated herein in their entirety.BACKGROUND

[0002] Amniotic fluid (AF) is a biologically complex fluid surrounding the developing fetus, providing essential mechanical protection, nutrients, and bioactive components critical for fetal development. This fluid has garnered increasing attention in regenerative medicine for its rich composition of proteins, peptides, growth factors, cytokines, and extracellular vesicles (EVs) that exhibit significant therapeutic potential. These molecular components play crucial roles in tissue repair, immune modulation, and cell signaling, making amniotic fluid a promising candidate for advanced medical applications, including wound healing, orthopedic repair, and neuro-regeneration.

[0003] Despite the biological potential of amniotic fluid, challenges remain in translating its components into effective clinical applications. This includes ensuring consistent decellularization and standardizing the isolation of therapeutic agents without compromising their bioactivity. Traditional processing methods may not sufficiently address these issues, limiting the scalability and efficacy of AF-based treatments. There is need for processing techniques to overcome these obstacles and unlock the full potential of amniotic fluid for research and clinical purposes.DETAILED DESCRIPTION

[0004] The present disclosure provides processing methods for amniotic fluid (AF) designed to optimize bioactivity retention and scalability for clinical and research applications. An object of the processes is to create a decellularized amniotic fluid composition with enhanced therapeutic potential, suitable for various regenerative treatments such as wound healing, orthopedic repair, and neuro-regeneration.

[0005] The composition of amniotic fluid evolves during gestation, transitioning from being initially composed of water and electrolytes to containing a wide variety of proteins, immunoglobulins, growth factors, as well as extracellular matrix components such as 14900-2485-6454\lglycosaminoglycans (GAGs) and collagens. Many enzymes, cytokines and growth factors found in AF have a number of therapeutic applications, such as in immunomodulation and regenerative therapies. A fraction of these bioactive molecules are solubilized or suspended in the fluid component of AF (termed herein the “soluble fraction’’). However, another fraction is contained within extracellular vesicles (EVs) secreted by or contained within cells found in amniotic fluid, which include both differentiated cells and mesenchymal and embryonic stem cells. However, the cells themselves are prone to senescence and therefore not amenable to culture. Furthermore, it is very difficult to store amniotic fluid for a significant time while maintaining the integrity of the amniotic fluid cells. The present disclosure describes methods of processing amniotic fluid to produce an amniotic fluid composition that includes bioactive components of amniotic fluid cells, yet is also acellular, i.e. lacking free of intact or ruptured cells or cellular debris.

[0006] In various embodiments, the methods can comprise obtaining amniotic fluid and subjecting a volume of the fluid to centrifugation to separate its solid and liquid components. In some embodiments, the amniotic fluid is centrifuged at a speed and for a time selected to produce a precipitate containing amniotic fluid cells and associated cellular material. In various embodiments, the centrifugation speed is from about 500 rpm to about 2500 rpm, for example about 500 rpm, about 600 rpm, about 700 rpm, about 800 rpm, about 900 rpm, about 1000 rpm, about 1100 rpm, about 1200 rpm, about 1300 rpm, about 1400 rpm, about 1500 rpm, about 1600 rpm, about 1700 rpm, about 1800 rpm, about 1900 rpm, about 2000 rpm, about 2100 rpm, about 2200 rpm, about 2300 rpm, about 2400 rpm, or about 2500 rpm. In particular embodiments, the centrifugation speed is from about 1000 rpm to about 2000 rpm.

[0007] The centrifugation time can be selected to accomplish the desired degree of separation in combination with the selected speed. In various embodiments, the centrifugation time can be from about 5 minutes to about 60 minutes, for example, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes. In particular embodiments, the centrifugation time is about 10 minutes to about 30 minutes. In an example embodiment, centrifugation is performed at about 1500 rpm for about 20 minutes. In certain embodiments, centrifugation is conducted at below room temperature (about 20 °C), for example at about 4 °C.

[0008] The centrifugation step yields an amniotic fluid supernatant that is largely free from cells but may still contain some residual cells, cellular debris and other large particulate matter. In various embodiments, ultrafiltration can be employed to remove these solids from the supernatant. More specifically, the amniotic fluid supernatant can be collected and subjected to 2490G-2485-6454Mat least two sequential stages of ultrafiltration configured to remove these residual solids to yield a decellularized amniotic fluid. Particularly, each stage of ultrafiltration can comprise biocompatible buffer solution. Suitable buffer solutions include, without limitation, phosphate buffered saline (PBS), 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid (HEPES) buffer, Tris-HCl buffer, glycine buffer, citric acid-sodium citrate buffer, and buffers comprising sucrose. In some cases, the composition of the buffer solution may be adjusted (e.g. reducing salt content) to make it sufficiently hypotonic. Other solutions suitable for inducing osmotic lysis include deionized water, ammonium chloride solution, or detergent (e.g. Triton X-100 or NP-40) in a low-salt buffer.

[0009] In preferred embodiments, the volume of resuspension solution is at or near the minimum that is sufficient for full resuspension of the precipitated material so as to produce a concentrated cell suspension. For example, the volume ratio of precipitate to resuspension solution can be about 0.5:1 to about 1:0.5. In particular embodiments, this ratio is about 1:1.

[0010] The method can further comprise incubating the mixture for a period of time to allow lysis and release to proceed. Gentle agitation may also be used to aid mixing of the solution with the precipitated material. In some embodiments the incubation time is about 5 minutes to about 60 minutes, or more particularly about 10 minutes to about 30 minutes. The cell suspension is then centrifuged to precipitate the cellular debris and yield an amniotic cell supernatant. Centrifugation of the cell suspension can be performed under conditions selected to effectively precipitate cellular debris while preserving the integrity of bioactive molecules. In some embodiments, the centrifugation speed is from about 500 rpm to about 2500 rpm, for example about 500 rpm, about 600 rpm, about 700 rpm, about 800 rpm. about 900 rpm, about 1000 rpm, about 1100 rpm, about 1200 rpm, about 1300 rpm, about 1400 rpm, about 1500 rpm, about 1600 rpm, about 1700 rpm, about 1800 rpm, about 1900 rpm, about 2000 rpm, about 2100 rpm, about 2200 rpm, about 2300 rpm, about 2400 rpm, or about 2500 rpm. In particular embodiments, the centrifugation speed is from about 1000 rpm to about 2000 rpm. In some embodiments, centrifugation is performed at a temperature selected to preserve the stability of bioactive elements, e.g. EVs and cytokines. In certain embodiments, centrifugation is conducted at below room temperature (about 20 °C), for example at about 4 °C.

[0011] The amniotic cell supernatant can then be filtered to yield an amniotic cell extract that is rich in bioactives such as EVs and cytokines. For example, filtration can comprise directing a flow of the amniotic cell supernatant to a separation membrane having an average effective pore size of about 0.1 to about 1 pm, for example, about 0.1 pm, about 0.2 pm, about 0.3 pm, about 0.4 pm, about 0.5 pm, about 0.6 pm, about 0.7 pm, about 0.8 pm, about 0.9 pm. or about 490G-2485-6454M1 pm. In some embodiments, stepwise filtration can be employed to enhance filtration efficiency. In some embodiments, stepwise filtration may comprise initial filtration with a membrane having a larger pore size to remove larger particulates and cellular debris, followed by filtration at one or more smaller pore sizes to ensure the removal of finer debris while retaining smaller bioactive molecules and EVs. For example, filtration of the amniotic cell supernatant can comprise one or more steps in which the fluid is initially passed through a membrane having a pore size greater than about 0.4 pm, such as about 0.5 pm to about 1 pm, then is filtered through a membrane having a pore size of about 0.2 pm or less, such as about 0.2 pm or about 0.1 pm. In particular embodiments, the final membrane in a plurality of filtering steps has a pore size that is smaller than all filters in previous steps. Filtration of the amniotic cell supernatant can be effected by TFF or NFF. In various embodiments, TFF may be preferred for larger volumes to reduce or prevent clogging and ensure process efficiency.

[0012] In various embodiments, the amniotic cell extract can be combined with the decellularized amniotic fluid to produce a decellularized amniotic fluid composition. In this way the composition can contain a representative spectrum of the bioactive molecules— the soluble fraction, vesicle-bound fraction and the previously cell-bound fraction— present in the original AF sample, while being substantially free of cells, cellular debris and other large particulates. In some embodiments, less of the decellularized amniotic fluid may be used in this step so as to increase the relative contribution of the previously cell-bound fraction to the total bioactives content in the composition. In some embodiments less of the amniotic cell extract may be used in this step so as to increase the relative contribution of the original soluble fraction to the total bioactives content in the composition. In certain embodiments, this step may comprise adding an amount of the amniotic cell extract that constitutes about 5% to about 50% of the decellularized amniotic fluid composition. The ratio of amniotic cell extract to decellularized amniotic fluid can be tailored based on the intended application. For example, a decellularized amniotic fluid composition for general use may comprise about 10% to about 25% amniotic cell extract and about 75 to about 90% decellularized amniotic fluid. For specialized applications that would benefit from higher concentrations of cell-derived bioactives (e.g.. cytokine-rich formulations), the contribution of the amniotic cell extract can be increased, for example up to about 40%, while maintaining sterility and molecular stability . Conversely, reducing the amount of amniotic cell extract can allow for a composition enriched in soluble and vesicle-bound fractions from the original fluid. This may be desirable for applications requiring lower immunogenicity or specific protein profiles.4490G-2485-6454M

[0013] The present disclosure provides a decellularized amniotic fluid composition containing a representative spectrum of the bioactive molecules present in the original AF sample, while being substantially free of cells, cellular debris and other large particulates. More specifically, the decellularized amniotic fluid composition can comprise bioactives including, but not limited to, proteins and peptides, EVs, cytokines, and growth factors. In various embodiments, the decellularized amniotic fluid composition can comprise peptides at a concentration of about 10 pg / ml to about 100 pg / ml, for example about 10 pg / ml, about 20 pg / ml, about 30 pg / ml, about 40 pg / ml, about 50 pg / ml, about 60 pg / ml, about 70 pg / ml, about 80 pg / ml, about 90 pg / ml, or about 100 pg / ml. In various embodiments, the decellularized amniotic fluid composition can comprise EVs at a concentration of about 1 x 108parti cl es / ml to about 1 x 1012particles / ml, for example about 1 x 108particles / ml. about 1 x io9particles / ml, about about 1 x io10particles / ml, about 1 x io11particles / ml. or about 1 x io12particles / ml. In various embodiments, the decellularized amniotic fluid composition can comprise cytokines and / or growth factors at a concentration of about 1 pg / ml to about 100 pg / ml for each individual factor, for example about 1 pg / ml, about 2 pg / ml, about 3 pg / ml, about 4 pg / ml, about 5 pg / ml, about 6 pg / ml, about 7 pg / ml, about 8 pg / ml, about 9 pg / ml. about 10 pg / ml. about 20 pg / ml, about 30 pg / ml, about 40 pg / ml, about 50 pg / ml, about 60 pg / ml, about 70 pg / ml, about 80 pg / ml, about 90 pg / ml, or about 100 pg / ml. In some embodiments, the growth factors include one or more of TGF-0 and VEGF. In some embodiments, the cytokines include one or more interleukins.

[0014] The processing methods described herein can further comprise preparing the decellularized amniotic fluid composition for long-term storage. Particularly, in accordance with the present disclosure, the composition can be stably stored for an extended period with little to no loss in bioactivity of its components. In various embodiments, storage comprises cry opreservation of the composition. In some embodiments, the composition can be divided into a number of single-use aliquots prior to cry opreservation. For example, a predetermined volume of the composition may be transferred into each of a plurality of sterile storage containers (e.g., cryogenic vials). The aliquot volume may be selected based upon the intended use; non-limiting examples include about 0.5 ml, about 1 ml. about 1.5 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, and about 10 ml.

[0015] Cryopreservation can further comprise placing the composition or aliquots thereof into a cryogenic storage unit and lowering the temperature to a selected long-term storage temperature. In some embodiments, the storage temperature is below about -50 °C, or more particularly about -75 °C to about -85 °C. In certain embodiments, the storage temperature is about -80 °C.5490G-2485-6454M

[0016] In some embodiments, the composition may be subject to a temperature reduction profile, in which the temperature is held for a time at at least one intermediate temperature before being lowered further to the storage temperature. In various embodiments the temperature reduction profile may include one or more intermediate temperatures selected from about -10 °C, about -15 °C, about -20 °C, about -25 °C, about -30 °C, about -35 °C, about -40 °C, about -45 °C, about -50 °C, about -55 °C, about -60 °C, about -65 °C, and about -70 °C, with the condition that each selected intermediate temperature is higher than the storage temperature. The stored composition may be held at each intermediate temperature for a time sufficient to allow each stored volume to reach a uniform state. In various embodiments, the composition may be held at an intermediate temperature for about 12 hours to about 48 hours, or more particularly for about 18 hours to about 30 hours.

[0017] In an aspect of the present disclosure, the above process is initiated with a sterile AF sample, and each step is performed so as to maintain sterility7. For example, AF collected by Cesarean section is typically obtained and packaged under strict sterile technique. Such a sample may be processed in accordance with the present disclosure under sterile conditions, including the use of sterilized vials, tubing, transfer vessels, and equipment for centrifugation, filtration, aliquoting, and cryopreservation. Various steps may also be performed under controlled temperature conditions, so as to maintain the subject material at temperatures that prevent or retard microbial growth. The process may further comprise quality control testing of the output of one or more processing steps to confirm sterility of the product.

[0018] Decellularized amniotic fluid compositions produced according to the present disclosure can be stored for an extended period with little to no loss in bioactivity. In some embodiments, a bioactivity of the decellularized amniotic fluid composition or a constituent molecule thereof is substantially unchanged by storage over a period of at least 9 months, or more particularly at least 12 months or at least 18 months. It is very difficult to store unprocessed amniotic fluid for a significant time while maintaining the integrity of the amniotic fluid cells. In contrast, the decellularized amniotic fluid composition of the present disclosure can exhibit this level of long-term without the addition of dimethyl sulfoxide (DMSO) or other penetrating or non-penetrating cry oprotectants.

[0019] The bioactive content of amniotic fluid can vary significantly between donor samples due to gestational age, maternal health status, and inherent biological variability. To mitigate or minimize such lot-to-lot variability7, the present disclosure provides methods comprising quantifying one or more characteristics of intermediate products during processing and adjusting6490G-2485-6454Mthe ratio of amniotic cell extract to decellularized amniotic fluid based on the quantified characteristics to produce a composition having a target value or falling within a target range.

[0020] The characteristic can be selected from, without limitation, protein concentration, peptide concentration, extracellular vesicle concentration, cytokine concentration, growth factor concentration, and a bioactivity measurement. Quantification can be performed using any suitable analytical technique, including spectrophotometric methods, colorimetric protein assays, particle counting methods, immunoassays, or functional bioactivity assays. In various embodiments, the method can comprise quantifying a characteristic at multiple stages during processing, including the raw amniotic fluid, the amniotic fluid supernatant following centrifugation, the cell suspension, the amniotic cell extract, or any combination thereof.

[0021] In an aspect of the present disclosure, the decellularized amniotic fluid and amniotic cell extract from a single donor can each be divided into portions and combined in different ratios to produce two or more compositions having different bioactive profiles. In some embodiments, the decellularized amniotic fluid is divided substantially equally while the amniotic cell extract is divided unequally, such that a higher-potency composition receives a greater proportion of the amniotic cell extract than a lower-potency composition.EXAMPLESExample 1: Decellularized amniotic fluid composition from a donor sample.

[0022] Amniotic fluid is processed by the following steps to produce a decellularized amniotic fluid:1. Sterile amniotic fluid collected from a donor is subjected to an initial centrifugation at 1500 RPM for 20 minutes at 4°C to separate cellular debris from the fluid. The supernatant is then collected and filtered through 0.2 pm and 0.1 pm ultrafiltration membranes in sequence. Post-filtration, a quality control sample is taken and analyzed to confirm sterility and cell-free status before proceeding to the next step.2. Next, 10 ml of precipitate is resuspended in 10 ml of IX PBS. The mixture is gently inverted to ensure uniform mixing and incubated at room temperature for 10 minutes. The resulting cell suspension is centrifuged and the supernatant is filtered, yielding a bioactive fluid rich in EVs and cytokines. This amniotic cell extract is combined with the decellularized amniotic fluid from the previous step.3. The filtered and processed decellularized amniotic fluid composition is divided into 10 mL aliquots, each labeled with a lot number and expiration date. The vials are placed in a 7490G-2485-6454Mcold storage container at -20 °C for 24 hours before being transferred to an ultralow freezer at -80 °C for long-term storage.Example 2: Standardized processing with mid-process quantification.

[0023] Sterile amniotic fluid from a single donor is processed to produce a decellularized amniotic fluid and amniotic cell extract according to Example 1. The decellularized amniotic fluid and the filtered amniotic cell extract are each subjected to spectrophotometric analysis. Based on the quantified protein concentrations, the combining ratio is calculated to achieve a target protein specification. The decellularized amniotic fluid and amniotic cell extract are combined according to the calculated ratio and cryopreserved.Example 3: Processing a single donor sample to yield decellularized amniotic fluid compositions having different bioactives profiles.

[0024] Sterile amniotic fluid from a single donor is processed to produce a decellularized amniotic fluid and amniotic cell extract according to Example 1. The decellularized amniotic fluid and the filtered amniotic cell extract are each subjected to spectrophotometric analysis. Based on the quantified protein concentrations, the combining ratios are calculated to achieve a number of target protein specifications. The decellularized amniotic fluid and amniotic cell extract are divided into portions and combined in different ratios to produce a plurality7of compositions having different concentrations of cell-derived bioactives.

[0025] Any methods disclosed herein comprise one or more steps or actions for performing the described method. The method steps and / or actions may be interchanged with one another. In other words, unless a specific order of steps or actions is required for proper operation of the embodiment, the order and / or use of specific steps and / or actions may be modified.

[0026] References to approximations are made throughout this specification, such as by use of the terms “substantially” and “about.” For each such reference, it is to be understood that, in some embodiments, the value, feature, or characteristic may be specified without approximation. For example, where qualifiers such as “about” and “substantially” are used, these terms include within their scope the qualified words in the absence of their qualifiers. All ranges also include both endpoints.

[0027] Similarly, in the above description of embodiments, various features are sometimes grouped together in a single embodiment or description thereof for the purpose of streamlining the disclosure. This method of disclosure, however, is not to be interpreted as reflecting an intention that any claim require more features than those expressly recited in that claim. Rather,8490G-2485-6454Mas the following claims reflect, inventive aspects lie in a combination of fewer than all features of any single foregoing disclosed embodiment.

[0028] The claims following this written disclosure are hereby expressly incorporated into the present written disclosure, with each claim standing on its own as a separate embodiment. This disclosure includes all permutations of the independent claims with their dependent claims. Moreover, additional embodiments capable of derivation from the independent and dependent claims that follow are also expressly incorporated into the present written description.

[0029] Without further elaboration, it is believed that one skilled in the art can use the preceding description to utilize the invention to its fullest extent. The claims and embodiments disclosed herein are to be construed as merely illustrative and exemplary, and not a limitation of the scope of the present disclosure in any way. It will be apparent to those having ordinary skill in the art, with the aid of the present disclosure, that changes may be made to the details of the abovedescribed embodiments without departing from the underlying principles of the disclosure herein. In other words, various modifications and improvements of the embodiments specifically disclosed in the description above are within the scope of the appended claims. Moreover, the order of the steps or actions of the methods disclosed herein may be changed by those skilled in the art without departing from the scope of the present disclosure. In other words, unless a specific order of steps or actions is required for proper operation of the embodiment, the order or use of specific steps or actions may be modified. The scope of the invention is therefore defined by the following claims and their equivalents.9490G-2485-6454M

Claims

CLAIMSWhat is claimed is:

1. A method for processing amniotic fluid, comprising:providing an amount of an amniotic fluid;centrifuging the amniotic fluid to produce an amniotic fluid supernatant and a precipitate comprising amniotic fluid cells;filtering the amniotic fluid supernatant in at least two sequential stages through separation membranes each having a pore size of about 1 pm to about 0.1 pm to produce a decellularized amniotic fluid;creating a cell suspension, comprising resuspending the precipitate in an aqueous solution selected to induce lysis of the amniotic fluid cells, wherein the volume ratio of precipitate to aqueous solution in the cell suspension is about 0.5:1 to about 1:0.5;centrifuging the cell suspension to produce an amniotic cell supernatant;filtering the amniotic cell supernatant to produce an amniotic cell extract; and combining the decellularized amniotic fluid and the amniotic cell extract to produce a decellularized amniotic fluid composition.

2. The method of claim 1. further comprising dividing the decellularized amniotic fluid composition into single-use aliquots.

3. The method of claim 1 or 2, further comprising lowering a temperature of the decellularized amniotic fluid composition to a storage temperature, wherein the storage temperature is below about -50 °C.

4. The method of claim 3, wherein the storage temperature is about -75 °C to about -85 °C.

5. The method of claim 4, wherein the storage temperature is about -80 °C.

6. The method of any one of claims 3 to 5, comprising storing the decellularized amniotic fluid composition at at least one intermediate temperature before storage at the storage temperature, wherein the at least one intermediate temperature is higher than the storage temperature.

7. The method of claim 6, wherein the at least one intermediate temperature is about -10 °C to about -75 °C104900-2485-6454\l8. The method of claim 7, wherein the at least one intermediate temperature is about -20 °C to about -40 °C.

9. The method of any one of claims 6 to 8, wherein the at least one intermediate temperature comprises a plurality of successively lower temperatures.

10. The method of any one of claims 6 to 9, comprising holding the decellularized amniotic fluid composition at the at least one intermediate temperature for about 12 hours to about 48 hours.

11. The method of claim 10, comprising holding the decellularized amniotic fluid composition at the at least one intermediate temperature for about 18 hours to about 30 hours.

12. The method of any one of claims 1 to 11, wherein the at least two sequential stages comprises:a first stage through a membrane having an average pore size of about 0.2 pm to about 1 pm; andone or more additional stages each through a membrane having an average pore size of about 0.1 pm to about 1 pm.

13. The method of claim 12, wherein the average pore size in the first stage is about 0.2 pm to about 0.5 pm.

14. The method of claim 12 or 13, wherein the average pore size in the one or more additional stages is about 0.1 pm to about 0.4 pm.

15. The method of any one of claims 12 to 14, wherein average pore size in the one or more additional stages is less than the pore size in the first stage.

16. The method of any one of claims 12 to 15, comprising 3 to 5 sequential stages.

17. The method of any one of claims 1 to 16, wherein the aqueous solution is a buffer solution selected from deionized water, phosphate buffered saline (PBS). 4-(2-hy droxy ethyl)- 1-piperazineethanesulfonic acid (HEPES) buffer, Tris-HCl buffer, and citric acid-sodium citrate buffer.

18. The method of claim 17, wherein the buffer solution includes one or more of sucrose, a detergent, and glycine.

19. The method of any one of claims 1 to 18, wherein the volume ratio of precipitate to aqueous solution in the cell suspension is about 1:1.11490G-2485-6454M20. The method of any one of claims 1 to 19, wherein resuspending comprises incubating the cell suspension for about 5 minutes to about 60 minutes.

21. The method of claim 20, wherein resuspending comprises incubating the cell suspension for about 5 minutes to about 15 minutes.

22. The method of any one of claims 1 to 21, wherein the first centrifuging step comprises a centrifugation time of about 5 minutes to about 60 minutes.

23. The method of claim 22, wherein the centrifugation time is about 10 minutes to about 30 minutes.

24. The method of any one of claims 1 to 23, wherein the first centrifuging step comprises a centrifugation speed of about 500 rpm to about 2500 rpm.

25. The method of claim 24, wherein the centrifugation speed is about 1000 rpm to about 2000 rpm.

26. The method of any one of claims 1 to 25. wherein combining comprises adding an amount of the amniotic cell extract that constitutes about 5% to about 50% of the decellularized amniotic fluid composition.

27. The method of claim 26, wherein the amount of the amniotic cell extract constitutes about 10% to about 25% of the decellularized amniotic fluid composition.

28. The method of any one of claims 1 to 27, wherein the second centrifuging step comprises a centrifugation speed of about 500 rpm to about 2500 rpm.

29. The method of claim 28, wherein the centrifugation speed is about 1000 rpm to about 2000 rpm.

30. The method of any one of claims 1 to 29, wherein the second filtering step comprises filtering the amniotic cell supernatant through at least one separation membrane having an average effective pore size of about 0.1 to about 1 pm.

31. The method of claim 30, wherein the second filtering step comprises filtering the amniotic cell supernatant through two or more separation membranes having successively smaller average effective pore sizes.12490G-2485-6454M32. The method of claim 31, wherein the second filtering step comprises filtering the amniotic cell supernatant through a first separation membrane having a pore size greater than about 0.4 pm, then through a second separation membrane having a pore size of about 0.2 pm or less.

33. The method of any one of claims 1 to 32, further comprising:quantifying a characteristic of the decellularized amniotic fluid, the amniotic cell extract, or both, prior to combining; andadjusting a ratio of the amniotic cell extract to the decellularized amniotic fluid based on the quantified characteristic to produce the decellularized amniotic fluid composition having a value for the characteristic within a target range.

34. The method of claim 33, wherein the characteristic is selected from protein concentration, peptide concentration, extracellular vesicle concentration, cytokine concentration, growth factor concentration, and bioactivity.

35. The method of claim 33 or 34, wherein quantifying comprises spectrophotometric analysis, colorimetric protein assay, particle counting, immunoassay, or functional bioactivity assay.

36. The method of any one of claims 33 to 35, further comprising quantifying the characteristic at one or more intermediate stages selected from: prior to the first centrifuging step, after the first centrifuging step, after creating the cell suspension, after incubating the cell suspension, after the second centrifuging step, and after the second filtering step.

37. The method of any one of claims 1 to 36, wherein each step is performed under sterile conditions.

38. An amniotic fluid product, comprising a decellularized amniotic fluid composition made by the method of any one of claims 1 to 37.

39. The amniotic fluid product of claim 38, wherein a bioactivity of the decellularized amniotic fluid composition is substantially unchanged by storage over a period of at least 9 months.

40. The amniotic fluid product of claim 39, wherein the period is at least 12 months.

41. The amniotic fluid product of claim 40, wherein the period is at least 18 months.

42. The amniotic fluid product of any one of claims 38 to 41, wherein the decellularized amniotic fluid composition is substantially free of any cryoprotectant.13490G-2485-6454M43. The amniotic fluid product of any one of claims 38 to 42, wherein the decellularized amniotic fluid composition comprise peptides at a concentration of about 10 pg / ml to about 100 pg / ml.

44. The amniotic fluid product of any one of claims 38 to 43, wherein the decellularized amniotic fluid composition comprises extracellular vesicles at a concentration of about 1 x 108parti cl es / ml to about 1 x io12parti cles / ml.

45. The amniotic fluid product of any one of claims 38 to 44, wherein the decellularized amniotic fluid composition comprises at least one cytokine at a concentration of about 1 pg / ml to about 100 pg / ml.

46. The amniotic fluid product of claim 45, wherein the at least one cytokine is an interleukin.

47. The amniotic fluid product of any one of claims 38 to 45, wherein the decellularized amniotic fluid composition comprises at least one growth factor at a concentration of about 1 pg / ml to about 100 pg / ml.

48. The amniotic fluid product of claim 47, wherein the at least one growth factor is selected from TGF-p and VEGF.

49. A method for processing amniotic fluid from a single donor to produce a plurality7of compositions having different bioactive profiles, comprising:providing an amount of an amniotic fluid from a single donor;centrifuging the amniotic fluid to produce an amniotic fluid supernatant and a precipitate comprising amniotic fluid cells;filtering the amniotic fluid supernatant to produce a decellularized amniotic fluid; resuspending the precipitate in an aqueous solution to create a cell suspension; centrifuging the cell suspension to produce an amniotic cell supernatant;filtering the amniotic cell supernatant to produce an amniotic cell extract;dividing the decellularized amniotic fluid into a plurality of portions;dividing the amniotic cell extract into a plurality of portions; andcombining the portions of decellularized amniotic fluid with the portions of amniotic cell extract in different ratios to produce a plurality of decellularized amniotic fluid compositions each having different concentrations of cell-derived bioactives.

50. The method of claim 49, wherein the decellularized amniotic fluid is divided into substantially equal portions and the amniotic cell extract is divided into unequal portions.14490G-2485-6454M51. The method of claim 49 or 50, further comprising quantifying a characteristic of the decellularized amniotic fluid, the amniotic cell extract, or both, prior to dividing or combining.

52. A plurality of decellularized amniotic fluid compositions made from amniotic fluid from a single donor by the method of any one of claims 49 to 51.15490G-2485-6454M