Methods and compositions related to reducing cell culture viscosity

Abstract

Hyaluronidase can be added to cell cultures to reduce viscosity. This invention relates to methods, compositions, and kits for adding hyaluronidase to said cell cultures.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 583,942, filed on September 20, 2023, which is incorporated herein by reference in its entirety. Background Technology

[0003] The basic environmental requirements for optimal cell growth in culture are: controlled temperature, suitable substrate for cell attachment, and appropriate growth medium and incubator that maintain the correct pH and osmotic pressure. The most important and critical step in cell culture is selecting a suitable growth medium for in vitro culture (Arora, M. Cell Culture Media: A Review. Material Methods 2013;3:175).

[0004] Mesenchymal stromal cells (MSCs) can be isolated from various tissues, such as bone marrow aspirate, fat, or cord blood. These cells possess the ability to proliferate and differentiate in vitro into a range of mesodermal cell lineages, including osteoblasts, chondrocytes, adipocytes, myocytes, and vascular cells. Due to this ability, MSCs provide an attractive source of progenitor cells for both research and clinical applications in tissue regeneration. A key factor for successful in vitro proliferation and differentiation of MSCs is the culture conditions (Vater, C. et al., Acta Biomaterialia, 2011; Vol. 7, No. 2, pp. 463-477).

[0005] The evolution of MSC culture from small-scale laboratory devices to large-scale bioreactors represents a significant milestone in biomanufacturing (Rowley, JA, Boychyn, M., Kelly, T. (2022) Why Cell Manufacturing Matters: How bioprocess innovations have laid the foundation for a cell-based products revolution, BioProc Intl, 20(10), 10-43). Traditionally, MSCs have been propagated on multiwell plates and other similar platforms, which, while effective for research and small-scale applications, are not suitable for commercial-scale production. The transition to bioreactor systems not only enables scalable growth of these cells under controlled, reproducible conditions but also conforms to current Good Manufacturing Practices (cGMP), which is crucial for therapeutic applications.

[0006] This shift is driven by the need for higher cell yields and more efficient manufacturing processes to meet the clinical and commercial demands of cell-based therapies. Bioreactors provide a controlled environment for precisely manipulating culture conditions such as oxygen, pH, and nutrient supply, which are crucial for maintaining the quality and function of MSCs. Furthermore, the use of bioreactors facilitates batch and perfusion cultures, thereby enhancing cell expansion capacity and product consistency.

[0007] These advances in MSC culture technology have not only optimized the production of MSCs themselves but have also paved the way for the production of MSC-derived extracellular vesicles (EVs). EVs (especially exosomes) are being explored for their therapeutic potential, possessing regenerative and immunomodulatory capabilities similar to their parent cells, but with reduced risks and better handling properties. Bioreactor systems originally developed for MSCs are now being adapted to support the production of MSC-EVs, enabling the generation of sufficient quantities of these potent therapeutic agents for clinical research and potential therapeutic uses.

[0008] However, scaling up MSC EV production to meet clinical demand presents a significant challenge. As MSCs begin to be used as the cell line for EV production, MSC culture needs to be scaled up in both time and size to maximize productivity (Adlerz, K., Patel, D., Rowley, JA, Ng. K., Ahsan, T. (2020) Strategies for Scalable Manufacturing and Translation of MSC-derived Extracellular Vesicles, StemCell Res, 48, 101978). A specific challenge is that MSC microcarrier suspension cultures become ineffective after a long period during EV production, and this can be exacerbated when cultured in serum-free media. The medium experiences a viscosity increase to the point where microcarriers aggregate and fall from the solution to the bottom of the bioreactor, essentially ending the culture. Addressing this phenomenon and minimizing viscosity increases allows for longer culture times and enables longer EV collections for industrial-scale production processes.

[0009] Processing and purifying exosomes from MSC conditioned media collected in flasks or bioreactors can be achieved through several methods, each with varying scalability and efficiency, which is crucial for transitioning from laboratory research to clinical and commercial production. This post-culture processing is often referred to as "downstream processing" in the pharmaceutical industry.

[0010] In small-scale settings, differential centrifugation is a common method for isolating exosomes. This process involves a series of centrifugation steps at progressively increasing speeds to remove cells and debris, followed by ultracentrifugation at even higher speeds (e.g., 100,000 g) to precipitate the exosomes. While effective for small volumes, this method is time-consuming and may be undesirable for maintaining the functional integrity of exosomes due to the high shear forces involved. Furthermore, differential ultracentrifugation typically produces low yields and may not be practical for scaling up to meet clinical needs.

[0011] For larger-scale and more efficient purification, scalable biopharmaceutical manufacturing technologies such as tangential flow filtration (TFF) and chromatography are preferred. Prior to the TFF stage, the culture medium must be clarified by filtration (typically multi-step filtration) to remove cell debris and contaminating particles that could contaminate the TFF filter, thus reducing clearance and yield. This clarification step can begin with a large-cutoff filter of several micrometers, followed by smaller filters (e.g., 0.8 micrometers, and then 0.45 micrometers). This clarified conditioned medium is then ready for efficient downstream purification.

[0012] TFF utilizes a cross-flow mechanism that allows for continuous processing of large volumes, effectively concentrating and percolating exosomes and other soluble proteins from process impurities. This method is advantageous due to its scalability, speed, and ability to maintain the bioactivity of exosomes, as it is gentler than ultracentrifugation. Chromatography (whether size exclusion chromatography or ion exchange chromatography) provides another scalable solution, enabling the separation of exosomes based on size and / or charge. A second TFF is typically performed after chromatography to finally percolate the product stream into a buffer and concentrate the solution. Then, a final sterile 0.2-micron filtration is performed before final bottling of the product. These methods are scalable and GMP-compatible. One challenge in concentrating MSC cell culture media via TFF is that the medium can become significantly more viscous, making it difficult to process, thus reducing EV yields and causing process failures due to high pressure, filter clogging, or contamination. Highly viscous solutions pose challenges to chromatographic purification, thus presenting a challenge to this emerging field of exosome therapy.

[0013] Hyaluronidase is an enzyme that plays a vital role in the mammalian body by breaking down hyaluronic acid (a major component of the extracellular matrix), thereby promoting tissue permeability and cell movement. This enzyme is involved in many physiological processes, including wound healing, tissue remodeling, and sperm penetration during fertilization. By degrading hyaluronic acid, hyaluronidase reduces the density of the extracellular matrix, allowing fluids to diffuse throughout the tissue and facilitating the physical migration of cells.

[0014] Hyaluronidase is widely used in both medical and cosmetic applications due to its ability to break down hyaluronic acid (a key component of the extracellular matrix). Its main uses include promoting subcutaneous fluid infusion (subcutaneous perfusion), enhancing drug absorption and dispersion in subcutaneous tissues, and promoting the absorption of contrast agents in urography (subcutaneous urography). In cosmetic surgery, hyaluronidase is crucial for dissolving hyaluronic acid fillers, especially in cases of complications or unsatisfactory results, and is also used to treat granulomatous foreign body reactions and manage skin necrosis associated with filler injections. These applications highlight the critical role of hyaluronidase in improving drug efficacy and resolving various medical and cosmetic surgical complications. (Jung et al., Arch Plast Surg 2020;47:297-300)

[0015] Hyaluronidase is widely used to isolate cells from various tissues because hyaluronic acid is a key structural component within the extracellular matrix, providing the physical structure of tissues. By adding HAase (with or without the addition of other ECM-degrading enzymes, such as collagenase or trypsin), researchers have been able to isolate healthy cells from tissues with optimized parameters. Human kidney cells can be obtained from kidney tissue using HAase as well as other enzymes such as collagenase (Yaigoub et al. 2022 Front. Cell Dev. Biol. 10:822275) and bovine articular cartilage to isolate chondrocytes using collagenase II (Mourik et al. Tissue Engineering Part C: Methods, Vol. 29, No. 1). In liver tissue, HAase facilitates the isolation of hepatocytes when it binds to collagenase in the perfusion medium (Drochmans et al. J. Cell Biol. 1975, Vol. 66, No. 1), while in Wharton's jelly from the umbilical cord, the HAase is part of a mixed enzyme-explant method for the extraction of mesenchymal stem cells (MSCs) (Azandeh et al. Journal of Biomedical Science and Engineering, Vol. 5, No. 10, October 2012). Hyaluronidase has been used in combination with other enzymes to isolate testicular cells, particularly germ cells, from testicular tissue (Yang et al. Reproduction, Fertility and Development 22(7) 1057-1065, 2010), and hyaluronidase has been used in combination with other digestive enzymes to isolate functional mast cells from bovine lung tissue (Stahl et al. Journal of Veterinary Medicine, Series B, Vol. 43, 1996).

[0016] HAases are also used in other areas where hyaluronic acid is used, such as in dermal fillers and tissue engineering. Injectable hyaluronic acid (HA) hydrogels have been popularized in facial aesthetics due to their long-lasting effects and low risk of complications. These fillers can be easily removed by HAase action (Perera GGG, Argenta DF, Caon T. The rheology of injectable hyaluronic acid hydrogels used as facial fillers: A review. Int J Biol Macromol. 2024 May;268(Part 2)). HA can be crosslinked to prepare tissue-engineered scaffolds (Sekar MP et al. ACS Biomater Sci Eng. 2023 June 12;9(6):3134-3159), and hyaluronidases are commonly used to degrade these scaffolds to study mechanical properties or to remove cells for evaluation (Wang et al. PLoS One. 2013 June 10;8(6):e56330).

[0017] Muller et al. (J Immunol Methods. 2014 Sep;411:55-65) tested HAase, as well as DNase and RNase, for the recovery of exosomes from human plasma, but concluded that "attempts to improve the recovery of exosomes from frozen / thawed plasma samples by enzymatic treatment are neither productive nor cost-effective and are not recommended," thus teaching a departure from this invention. Previous use of HAase in cell culture has primarily revolved around decomposing tissue ECM to isolate cells or decomposing cross-linked HA to study the mechanical properties of the underlying material.

[0018] The article “Hyaluronic Acid: An Overlooked Extracellular Vesicle Contaminant” by Goncalves et al. (2023) highlights the presence of hyaluronic acid (HA) as a previously underestimated contaminant in extracellular vesicle (EV) samples. They discuss how HA (a polysaccharide released by cells of various vertebrates) can contaminate EV samples, particularly when using separation methods such as size exclusion chromatography and tangential flow filtration. The study shows that using these filtration-based separation methods, medium and low molecular weight HA polymers are inadvertently retained in the EV fraction, resulting in HA contamination in the sample. The study demonstrates that while these methods are effective in removing non-EV-related proteins, they do not completely eliminate HA. For example, medium molecular weight HA (289 kDa) was only partially removed, with a significant amount retained in the final EV fraction. This retention varies depending on the molecular weight of HA and the separation technique used, suggesting that HA contaminants may inadvertently co-separate with EVs, thus affecting the purity and potential sensing bioactivity of EV samples. Although this manuscript describes HA as a contaminant for MSC-EV formulations, it does not discuss how this may affect cell culture processes or purification yields.

[0019] In PCT application “WO2018208670A1”, it is described that extracellular vesicles (EVs) such as those derived from umbilical cord, placental tissue, or mesenchymal stem cells (MSCs) are covered by a complex of hyaluronic acid and proteoglycans, and that the EVs can adhere together. Hyaluronidase is used to treat the EVs during processing (at a concentration of 1500 U / mL) to produce a single EV formulation, thereby removing the hyaluronic acid and proteoglycan complex from the surface, thus exposing tethered TGF-β for enhanced therapeutic use. It is also believed that HAase treatment enhances the separation and purification of extracellular vesicles (EVs) having transforming growth factor-β (TGF-β) tethered to their membrane surface by removing the hyaluronic acid and proteoglycan complex from the surface of the EVs during centrifugation. WO2018208670A1 only teaches about hyaluronic acid on the EV surface and does not mention increasing yield during filtration or other pharmaceutical processing steps other than centrifugation.

[0020] PCT application “WO 2017 / 151862 A1” discloses matrix-bound nanovesicles and their uses. This PCT demonstrates that EVs can be isolated from extracellular matrix (ECM) materials derived from various tissues, such as bladder matrix (UBM), submucosal layer of small intestine (SIS), or other suitable tissues. Various enzymes are used to process the ECM to isolate EVs from it, but not for cell culture or purification applications.

[0021] The reference “Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles” by Boerre et al. (J. Extracellular Ves, 2016) investigated the optimization of hyaluronidase (HYase, also referred to herein as HAase) pretreatment for the isolation of extracellular vesicles (EVs) from synovial fluid (SF). This reference focuses on using hyaluronidase to facilitate the isolation of EVs from synovial fluid. Existing techniques address the challenge of the high viscosity of synovial fluid due to the presence of hyaluronic acid (HA), which hinders EV isolation during ultracentrifugation. In this context, hyaluronidase is used to degrade HA and improve EV recovery, particularly the recovery of CD44+ from this naturally occurring biofluid. + Vesicles. The methods outlined in the prior art do not address the specific challenges or requirements associated with the industrial production of cell culture, bioreactor environments, or EVs.

[0022] The industrial production of EVs requires scalable production technologies (such as bioreactors) and scalable purification systems (such as filtration, TFF, and chromatography). Current technologies lack methods for extending the culture time of EV-producing cells in bioreactors (e.g., by minimizing viscosity accumulation over time). These scalably produced EVs also require purification using conventional biopharmaceutical techniques. Achieving scalable production and purification of EVs allows for the production of larger quantities of product, recovery of more product during purification, fewer process failures, and greater overall manufacturability, thereby reducing the cost per unit volume of the product. Summary of the Invention

[0023] This article discloses a method for improving the properties of liquid cell culture media, the method comprising introducing hyaluronidase (HAase) into the culture medium.

[0024] This article also discloses a method for improving the properties of a liquid cell culture medium, the method comprising culturing cells in the liquid culture medium, removing the cells, and then introducing HAase into the liquid culture medium after cell removal.

[0025] This article further discloses a composition comprising liquid cell culture medium and hyaluronidase.

[0026] This article also discloses a kit containing liquid cell culture medium and hyaluronidase.

[0027] This document further discloses a composition comprising a conditioned medium derived from MSC cell cultures, wherein the conditioned medium contains EVs, wherein the presence of EVs is between 1e8 and 1e11, and further wherein the conditioned medium contains exogenously added HAase at a concentration of about 0.01 U / mL to 100 U / mL, wherein the conditioned medium has not undergone clarification or filtration. Attached Figure Description

[0028] Figures 1A-B A typical EV production test run is illustrated, where microcarrier aggregation and MSC culture contamination can be disrupted under selective medium and bioreactor conditions (i.e., EV collection medium B). Figure 1A illustrates a simplified upstream bioprocess protocol where single-vial MSCs are first amplified on 2D T-225 CellBIND flasks, and then, on day 0 of EV preparation, these single-vial MSCs provide inoculum material for culture on microcarriers in an Ambr250® culture vessel. After sufficient amplification, the RoosterNourish medium is replaced on day 5 with a medium specifically formulated for the EV collection phase (A or B). Figure 1B shows photographic images of the time process in a representative Ambr250® culture bioreactor containing live hMSCs. The image from day 5 demonstrates the effect of switching from RoosterNourish amplification medium to EV collection medium (A, B, and B+ dextran sulfate). Microcarrier aggregation and contamination of MSC cultures with collection medium B or even B+ dextran sulfate fail to reduce clumping or offset the loss of EV-conditioning medium as expected. Cells maintained in culture medium A did not show significant microcarrier aggregation, while microcarriers containing cells in culture medium B continued to aggregate and precipitate to the bottom of the flask, contaminating the culture by day 10.

[0029] Figure 2 The study showed a sharp increase in viscosity index in cells maintained in EV collection medium B at day 10. High medium viscosity presents unique challenges for microcarrier cell culture and for the collection of secreted cell culture products. This phenomenon is accompanied by culture contamination of cells grown on the microcarriers. Cell culture viscosity was measured and recorded as a viscosity index, or [(viscosity-time)]. 样品 (Viscosity time) 水对照 )] / (viscosity time 水对照 Viscosity time refers to the total migration time of 1 mL of liquid solution from the syringe attached to the ring support through a standard 21-gauge blunt needle. The viscosity index is controlled to account for different relative humidity and atmospheric conditions.

[0030] Figure 3A -C shows the effect of hyaluronidase on culture viscosity.Figure 3A Treatment with 500 mU / mL hyaluronidase (HYAL) [Bovine HYAL (NP_001017941.1; 1.0 mU / mL, MilliporeSigma, Saint Louis, Missouri)] showed that treatment could selectively improve post-collection culture medium viscosity compared to collagenase, Accumax, or TrypLE, which had no effect on viscosity index. Figure 3B The dose response of HYAL (0 U / mL to 100 U / mL) to the viscosity index of MSC conditioned medium was demonstrated compared with TyrpLE and trypsin. Figure 3C It was verified that treating MSC conditioned medium in a range of HYAL concentrations or cell isolation formulations (1x TrypLE, 1x trypsin) did not affect EV particle counts as measured by NTA (nanoparticle tracking analysis).

[0031] Figure 4 The dose-response relationship between the concentration of recombinant HIS-labeled human HYAL (NP_001167515.1; 0.1 mU / mL, 1.0 mU / mL, or 10 mU / mL, ACRO; rPH20 from ACRO Biosystems, Newark, Delaware) in the EV-conditioning collection medium after addition on day 7 is shown to the viscosity index compared to bovine HYAL (NP_001017941.1; 1.0 mU / mL, MilliporeSigma, St. Louis, Missouri) or untreated. The comparability of per-unit activity between the human recombinant isoform of HYAL and the bovine testicular isoform was depicted by the viscosity index collected on days 10 and 12.

[0032] Figure 5 It was shown that even at concentrations up to 500 U / ml, hyaluronidase did not appear to have a negative impact on MSC morphology / viability during flask culture, and the cells remained adherent to the culture surface.

[0033] Figure 6A -B shows that cells detached from the microcarrier when 250 U / ml of hyaluronidase was used. Figure 6A and 6B The proliferation of cells with and without hyaluronidase treatment is shown.

[0034] Figure 7A -C indicates that methods involving the addition of HYAL may require special optimization between variables related to the HYAL dosage and culture conditions. Figure 7AImages of labeled hMSCs attached to microcarriers (mc) grown in a microbioreactor are shown between day 3 and day 7 post-transfer of seed culture at different concentrations of HYAL. MSFv15 is a unique cell culture medium formulation. All the HYAL concentrations shown (10 U / mL to 250 U / mL) demonstrated that these concentrations did not support healthy cell cultures, and cells no longer attached to the microcarriers, resulting in poor cell growth. Figure 7B Images of healthy cultures at HYAL concentrations reduced to 0.001 U / mL (1 mU / mL) are shown, as can be seen from the fact that labeled hMSCs maintained attachment to microcarriers (mc) grown in a microbioreactor at a “microdose” of 1.0 mU / mL (- / +) HYAL between days 3 and 12 post-seed culture transfer. Figure 7C By from Figure 7B The viscosity index of the conditioned medium in the cell collection shown demonstrates the activity of HYAL. A dose of 1.0 mU / mL HYAL is sufficient to reduce the viscosity of the medium and allow hMSCs to attach under these culture conditions.

[0035] Figure 8 The study showed that adding hyaluronidase every 3 days still caused some cells to detach from the carrier.

[0036] Figure 9 The results showed that the viscosity of samples treated with hyaluronidase (both collected cells and TFF-concentrated cells) was significantly reduced.

[0037] Figure 10A -C demonstrates that treating exemplary adherent mammalian cells (hMSCs) in 2D planar cultures with varying doses of hyaluronidase (10-250 U / mL HYAL) (including very high doses (250 U / mL)) does not damage the cells throughout the treatment time. Figure 10A A representative experiment used to determine the toxicity of hyaluronidase to MSCs in culture is shown. HYAL was added to the cells on day 3 post-inoculation, and cell health was monitored by morphology (images at 10x magnification) until day 7. Viscosity index analysis of waste culture medium was performed concurrently with the validation of HYAL activity. Hyaluronidase levels tested were 0 U / ml, 10 U / ml, 30 U / ml, 100 U / ml, and 250 U / ml. It was found that even at concentrations up to 250 U / ml, hyaluronidase had no negative impact on MSC morphology / viability during flask culture, and the cells remained adherent to the culture surface. Figure 10B Depicting Figure 5The increased cell density shown in Figure A within the HYAL dose range on days 3 and 5 indicates that HYAL has no effect on growth even at a concentration of 250 U / mL. Figure 10C The maintenance of cell viability % at day 3 and day 5, as shown in Figure 11A, indicates that HYAL has no effect on viability even at a concentration of 250 U / mL.

[0038] Figures 11A-B Representative experiments are shown where treatment of EV conditioned medium with a wide range of HYAL concentrations (0 U / mL to 100 U / mL) did not adversely affect its bioactivity in cell culture assays for wound healing (Fig. 11A) or angiogenesis (Fig. 11B). CM samples were clarified, treated with HYAL, and ultracentrifuged. The EV precipitate was resuspended in assay medium, and the same number of particles were administered in each assay. More specifically, 14A shows the potency of EVs derived from day 10 hMSC microcarrier cultures and subsequently treated with a series of HYAL concentrations (0.0 U / mL to 100 U / mL) or trypsin (Tryp1x). Enzyme-treated EVs were applied to scratched HUVEC monolayers, and scratch closure was measured. Note the effect of trypsin on reduced wound closure activity and the lack of HYAL-related inhibition of EV bioactivity. (Strip, SD). Figure 11B shows representative images and quantitative angiogenic potency of day 10 hMSC microcarrier cultures derived from Ambr250 and subsequently treated with a series of HYAL concentrations (0.1 mU / mL to 100 U / mL) or trypsin. EVs were applied to HUVECs on a Cultrex gel layer and incubated overnight. Total mesh area was analyzed using an angiogenesis analyzer in ImageJ. It should be noted that active HYAL concentrations (0.0001 U / mL to 100 U / mL) did not impair the angiogenic mesh-forming ability of HUVECs. (Strips, SD)

[0039] Figure 12A -B demonstrates that cells grown in the Ambr bioreactor on microcarriers can be protected from culture contamination by timely addition of specific “micro-dose” concentrations of hyaluronidase (HYAL) down to 1.0 mU U / mL. Figure 12A The upstream EV bioprocess flow diagram is shown, in which MSCs are grown in Ambr250® flasks to optimize HYAL concentration and bioreactor physical process parameters. Figure 12BRepresentative images of the dose- and time progression of hMSC microcarrier cultures in Ambr250(r) flasks are shown, with day 5 being 5 days after seed culture inoculation into flasks containing microcarriers, at which point the amplification medium was exchanged with the EV collection medium. On day 6, different concentrations of HYAL (0.01 mU / mL; 0.1 mU / mL; 1 mU / mL) were added to the cultures. At concentrations < 1.0 mU / mL, microcarrier precipitation and culture contamination were observed on days 7–10, but no visible microcarrier aggregation and contamination were observed at the higher doses of 1 mU / mL (0.001 U / mL).

[0040] Figures 13A-C This study depicts how HYAL supplementation at concentrations between 0.01 mU / mL and 1.0 mU / mL, considered “micro-dose,” affects culture viscosity and yield. When hMSCs were cultured in microcarrier flasks with an active, optimized concentration of HYAL (1.0 mU / mL), yield was enhanced, and the total particle size and distribution of secreted cellular EV / exosome products were maintained. Figure 13A demonstrates that HYAL applied at the “optimal” concentration (1.0 mU / mL) was sufficient to rapidly reduce the viscosity of the EV collection medium, which contrasts with the previously observed reversal of microcarrier culture contamination. Figure 8 Consistent with the results. (Strip, SD). Figure 13B shows that the yield of cell products (extracellular vesicles, EVs) secreted into the conditioned medium was increased by approximately 3-fold after treatment with optimal HYAL (1.0 mU / mL) on day 6. Figure 13C shows the particle size distribution of EVs collected from this experiment from the conditioned medium, which is almost identical to the median size and distribution determined by analysis of the samples using a NanoSight NTA instrument.

[0041] Figures 14A-C show a series of stirring conditions tested in a rotary flask. Figure 14A shows a single condition tested in Ambr250. Figure 14B shows the effectiveness of the HYAL treatment under a series of stirring conditions. Over-stirring (6800 W / m) 3 This affects cell growth and attachment, and therefore the culture medium does not become viscous. Data were collected on day 10 of culture. NM = Not measured. 14C shows cell attachment under the conditions in Figure 14B. Cells were at 1.6–36 W / m². 3 Adhesion remains within the specified range, regardless of HYAL treatment. NM = Not measured.

[0042] Figures 15A-BAn experiment is shown that determined the appropriate HYAL concentration, temperature, and incubation time for treatment with conditioned medium (15LSTR) produced on a large scale. Figure 15A shows a 200 mL sample of conditioned medium collected after treatment with 100 mU / mL HYAL for approximately 10 minutes at room temperature, but incubation at 37°C, resulting in a decrease in viscosity. Surprisingly, HYAL appears to be active not only during cell culture incubation at 37°C but also at room temperature. Error bars are SD. Figure 15B shows the results from conditioned medium collected from the main 15L sample treated with 100 mU / mL HYAL for 10 minutes at room temperature. Error bars are SD.

[0043] Figures 16A-D An experimental bioprocess is illustrated in which active HYAL (- / +) can be used to reduce the viscosity of EV collection medium after cell culture and collection, thus demonstrating that HYAL (100 mU / mL × 30 min, room temperature) can improve various aspects of downstream processing (DSP) of secreted exosomes / EV cell products. 16A depicts the DSP protocol and compares it with a test 1.2 L volume of hMSC conditioned bioreactor medium (CM) treated with HYAL 100 mU / mL (- / +). 16B shows that HYAL treatment of CM reduces viscosity by approximately 4-fold. 16C and 16D show that HYAL treatment does not affect EV particle concentration (16C) and size distribution (16D).

[0044] Figures 17A-B The filtration of treated and untreated samples is shown. Figure 17A shows a large-pore depth filter, comparing HYAL-treated (0.1 U / mL) and untreated conditioned medium subjected to filtration 1. The untreated sample over-pressurized the filter, requiring replacement at a higher cost. The HYAL-treated sample did not experience this problem. Figure 17B shows a small-pore depth filter, comparing HYAL-treated (- / +) conditioned medium subjected to filtration 2 before chromatography. The untreated sample over-pressurized the filter, requiring replacement with a new filter at a higher cost, and operating at a lower flow rate, extending the processing time to >15 minutes. The HYAL-treated sample did not experience this problem, with filtration completed in approximately 5 minutes.

[0045] Figures 18A-CShows the cumulative particle recovery in samples treated with HYAL. Figure 18A shows that HYAL treatment according to the DSP protocol significantly increased the process recovery % of EVs (on a cumulative basis), and at the end of the process, 52% (HYAL-treated) was recovered compared to 4% (untreated). Figure 18B shows that the in vitro scratch wound closure activity of EVs was maintained after downstream processing with and without HYAL treatment. Experiments were performed using monolayer HUVEC cells. Control samples were either basal medium (negative control) or complete medium (positive control). Experimental samples were collected after the filtration 3 (final) step of DSP. Bars represent the mean of N = 6 (negative control), N = 4 (untreated), N = 7 (positive control, HYAL-treated) replicates performed for each group. Error bars represent SD. *, p < 0.05, analyzed by ANOVA and Dunnett's multiple comparison test. Figure 18C shows that the enzymatic activity of CD73 present on EVs was maintained after downstream processing with and without HYAL treatment. (Left) Assay controls included untreated (positive control) or heat-inactivated (negative control) recombinant human CD73 (rhCD73). (Right) Untreated and HYAL-treated samples (Figure 18A) were tested after the filtration 3 step. Bars represent the mean of N = 2 experiments performed for each group.

[0046] Figures 19A-C Shows the maintenance of the expression of EV markers CD63 (A), CD9 (B), and CD81 (C) before and after treatment with HYAL. Western blot results were obtained using the ProteinSimple Jess capillary western blot system. Samples were collected after the filtration 3 (final) step of DSP.

[0047] Figures 20A-B Shows that treatment with hyaluronidase (also referred to herein as "Agent V") minimizes pressure build-up during filtration.

[0048] Figure 21 Shows that hyaluronidase increased the particle yield by clarification.

[0049] Figure 22 Shows that hyaluronidase enables the use of a smaller filter cut-off (0.45 µm), thereby increasing the purification capacity.

[0050] Figure 23 Shows that treating conditioned medium with Agent V maintains EV quality attributes. For example, the expression of the tetraspanin markers is maintained.

[0051] Figures 24A and 24B show that the pressure improvement increases with increasing dose of agent V (hyaluronidase), reaching a saturation point of approximately 0.5 U / mL. Maximum yield benefit was observed at 1.0 U / mL. (A) shows the loading pressure, and (B) shows the particle yield.

[0052] Figures 25A-B The pressure curve was shown to improve the incubation time by increasing it to 30 minutes. Filtration was improved even with immediate addition of reagent V. Under these treatment conditions, the yield benefit saturated.

[0053] Figures 26A-B Temperature information is shown at 4°C, room temperature (RT), and 37°C. (A) shows the pressure, and (B) shows the particle yield.

[0054] Figures 27A-D The results show that after filtration, the size distribution does not exhibit a clear trend based on the treatment parameters of agent V. (A) Process; (B) Dose response; (C) Incubation time; (D) Temperature.

[0055] Figure 28 The pressure and loading results for samples treated with hyaluronidase (drug V) and untreated samples are shown.

[0056] Figures 29A-B The particle yield results for the treated and untreated samples are shown. (A) shows the particle concentration; (B) shows the particle yield.

[0057] Figure 30 The addition of hyaluronidase during downstream processing is shown.

[0058] Figure 31 An experimental design is shown that uses hyaluronidase to test human skin fibroblasts to determine the effectiveness of hyaluronidase in reducing viscosity and significant microcarrier clumping.

[0059] Figure 32 The images show that HDF microcarriers are largely aggregated, even more so than MSC cultures. 4x representative images.

[0060] Figures 33A-D The high cell count and high viscosity of EVs produced in HDF are shown. The culture is more viscous than MSC culture. (A) shows the sampled cell density. (B) shows particles / mL. (C) shows the viscosity index. (D) shows the frequency.

[0061] Figure 34 The following describes the post-collection processing of HDF using hyaluronidase titration.

[0062] Figures 35A-BThe viscosity treatment after HDF collection is shown. (A) Viscosity index at different concentrations of hyaluronidase is shown. (B) Percentage of EV recovered during filter testing is shown.

[0063] Figure 36 An experimental design for determining whether HEK cell exposure to hyaluronidase can affect EV yield is shown.

[0064] Figures 37A-B The results show that HEK medium is not viscous, but EV recovery still significantly increased from about 73% to about 100%. (A) Viscosity index of control and conditioned media is shown. (B) Filter test and particle recovery percentage of EV using different concentrations of hyaluronidase are shown. Detailed Implementation

[0065] General definition

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0067] In this document, a range may be expressed as from “about” a specific value and / or to “about” another specific value. “About” means within 10% of said value, for example, within 9%, 8%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of said value. When expressing such a range, the other side includes from said one specific value and / or to said other specific value. Similarly, when a value is expressed as an approximation using the antecedent “about”, it should be understood that said specific value forms the other side. It should be further understood that each endpoint of a range is significant relative to and independent of the other endpoint. It should also be understood that many values ​​are disclosed herein, and each value is disclosed herein as “about” the specific value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed.

[0068] As used herein, the term "comprise" and its variations are used synonymously with the term "comprising" and its variations, and are open, non-limiting terms. While the terms "comprise" and "comprising" have been used herein to describe various embodiments, the terms "consistently comprising" and "comprises" may be used in place of "comprise" and "comprising" to provide more specific embodiments, and are also disclosed. Throughout the description and claims of this specification, the word "comprise" and other forms of the word, such as "comprising" and "comprises," mean "including but not limited to," and are not intended to exclude, for example, other additives, components, wholes, or steps.

[0069] As used in the specification and claims, the singular forms “a,” “an,” and “the” include the plural objects unless the context clearly indicates otherwise. For example, the term “pharmaceutical” includes a variety of pharmaceuticals, including mixtures thereof.

[0070] As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and mean both the occurrence of the condition and the absence of the condition. Thus, for example, the statement that a formulation “may include excipients” means both the case where the formulation includes excipients and the case where the formulation does not include excipients.

[0071] The term "extracellular material" (also known as "EM") refers to cell-derived material, including extracellular vesicles (EVs) present in eukaryotic fluids, including conditioned media of blood, lymph, urine, saliva, and cell cultures. This extracellular material can be derived from endosomes and plasma membranes and can contain secreted components. Examples of these components include, but are not limited to, nucleic acids (such as DNA, RNA (including miRNA)), proteins, peptides, carbohydrates, lipids, and small molecules. Extracellular material can be cell-derived small vesicles and can include exosomes, microvesicles, extranuclear granules, exfoliated vesicles, membrane vesicles, plasma membrane vesicles or other membrane-bound assemblies, as well as apoptotic bodies, viruses, and cells. In some embodiments, the extracellular material may comprise, consist of, or be substantially composed of extracellular vesicles.

[0072] Unless otherwise specified, the term "cell" as used herein also refers to a single cell, cell line, primary culture, or culture derived from such cells. "Culture" is a composition containing isolated cells of the same or different types. Live cells can be found in cell cultures.

[0073] The term "mammalian cell" means any cell that is derived from or originates from any mammal (e.g., human, hamster, mouse, green monkey, rat, pig, cow, or rabbit). In some embodiments, the mammalian cell may be, for example, an immortalized cell, a differentiated cell, or an undifferentiated cell.

[0074] The term "pluripotent stem cell" refers to a cell capable of producing multiple different cell types. The term "mesenchymal stem cell" refers to a stem cell originally derived from mesenchyme. The term refers to a cell capable of differentiating into at least two or more of osteoblasts, chondrocytes, adipocytes, or myocytes. Mesenchymal stem cells (MSCs) are isolated from any type of adult tissue. Typically, MSCs are isolated from bone marrow, adipose tissue, umbilical cord, or peripheral blood. In a preferred aspect, MSCs are obtained from bone marrow or fat aspirates obtained from adipose tissue. The terms "multipotent" or "pluripotent" also encompass induced multipotent stem cells or induced pluripotent stem cells, or cells that have been induced to a pluripotent stage using any chemical or genetic means. In some embodiments, the multipotent stem cells or pluripotent stem cells of this disclosure are mesenchymal stem cells.

[0075] The term "population" as used in reference to the pluripotent stem cells and mesenchymal stem cells described herein refers to a group of cells that are molecularly identical or highly similar. Populations can be isolated from human tissues or can be grown and expanded in cultures. Any population described herein can be composed "substantially" of the cells described, such as pluripotent stem cells or mesenchymal stem cells. As used herein, "substantially" means that, from the perspective of cellular composition, the cell population is substantially pure. For example, the cells can be more than about 80%, 90%, 95%, 97%, 98%, or 99% pure.

[0076] The terms “unstimulated” or “primary” refer to a population of cells that are untreated, unpolarized, or not induced by the methods of this disclosure. Fresh or frozen primary isolated mesenchymal stem cells are considered unstimulated or primary.

[0077] A "control" is an alternative subject or sample used for comparative purposes in an experiment. A control can be "positive" or "negative".

[0078] The term “substantially free” means a composition (e.g., liquid culture medium) that is at least or about 90% free of a specific substance (e.g., mammalian cells or microcarriers).

[0079] The term “culture” or “cell culture” refers to the maintenance or growth of mammalian cells in a liquid culture medium under controlled physical conditions.

[0080] The term "liquid culture medium" refers to a fluid containing sufficient nutrients to allow mammalian cells to grow in vitro. For example, a liquid culture medium may contain one or more of the following: amino acids (e.g., 20 amino acids), purines (e.g., hypoxanthine), pyrimidines (e.g., thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, nicotinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc, selenium, and other necessary trace metals, as well as sodium bicarbonate. Liquid culture media may contain serum from mammals. In some cases, liquid culture media do not contain serum or other extracts from mammals (limited to liquid culture media). Liquid culture media may contain trace metals, mammalian growth hormones, and / or mammalian growth factors. Non-limiting examples of liquid culture media are described herein, and further examples are known in the art and are commercially available.

[0081] The term "microcarrier" refers to particles (e.g., organic polymers) having a size between 20 µm and about 1000 µm that contain a surface that allows or facilitates the attachment of mammalian cells (e.g., any mammalian cells described herein or known in the art). Microcarriers may contain one or more pores (e.g., pores having an average diameter of about 10 µm to about 100 µm). Examples of microcarriers are known in the art. For example, microcarriers may contain, for example, polymers (e.g., cellulose, polyethylene glycol, or poly-(lactic-co-glycolic acid)).

[0082] The term "animal-free liquid culture medium" refers to a liquid culture medium that does not contain any animal-derived components (e.g., proteins or serum).

[0083] The term "serum-free liquid culture medium" refers to a liquid culture medium that does not contain animal serum.

[0084] The term "solid culture medium containing serum" refers to a liquid culture medium containing animal serum.

[0085] The term "chemically defined liquid culture medium" refers to a liquid culture medium in which virtually all chemical components are known. For example, chemically defined liquid cultures do not contain fetal bovine serum, purified bovine serum albumin, or purified human serum albumin, because these preparations are typically isolated from blood and contain a complex mixture of albumin and lipids. Chemically defined media may contain recombinant albumin.

[0086] The term "protein-free liquid culture medium" refers to a liquid culture medium that does not contain any protein (e.g., any detectable protein).

[0087] Viscosity is the magnitude of internal friction within a fluid, specifically the intermolecular friction between adjacent particles moving at different speeds. The magnitude, shape, and amplitude of the intermolecular forces within a fluid determine its viscosity. As cell cultures proliferate, metabolic activity and other processes may be expected to increase the number of solute particles in the culture medium, thereby increasing its viscosity. The viscosity of cell cultures is measured and recorded as a viscosity index, or [(viscosity-time)]. 样品 (Viscosity time) 水对照 )] / (viscosity time 水对照 Viscosity time refers to the total migration time of 1 mL of liquid solution from the syringe attached to the ring support through a standard 21-gauge blunt needle. The viscosity index is controlled to account for different relative humidity and atmospheric conditions.

[0088] Composition, kit and method of use

[0089] This document discloses methods, kits, and compositions for extending the lifespan of cell cultures by reducing the viscosity of cell culture media, thereby making cell culture more productive. Methods, kits, and compositions for improving the quality of culture media, thereby enabling easier and higher-yield purification of biological products (such as EVs) using the media, are also disclosed. The quality of the culture media can be improved in terms of yield, quality, or processing efficiency. This improvement can be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 10-fold or more, compared to a control under the same conditions but without HAase.

[0090] The quality of a cell culture medium can be improved by introducing hyaluronidase before, during (simultaneously with), or after culturing. Hyaluronidase can be added to cell culture media in various culture configurations, or even after cells have been removed from the medium for purification processes. For example, cells can be grown on microcarriers in a bioreactor configuration or in flasks. The invention also relates to improving the purification of cell culture media containing EVs (e.g., EVs already grown in a bioreactor or flask) by reducing pressure buildup during filtration, thereby achieving higher yields. These and other uses will be discussed in detail below.

[0091] As used herein, “hyaluronidase” refers to a class of hyaluronic acid-degrading enzymes. Hyaluronidases used with this invention may be naturally occurring or recombinant. They may be synthetically prepared or naturally derived. It is noteworthy that hyaluronidase may also be referred to herein as “HAase,” “HYAL,” or “Pharmaceutical V.” All of these are defined as identical to “hyaluronidase” as defined in detail below. Those skilled in the art will understand how to obtain and use hyaluronidases through the methods and compositions disclosed herein. Examples of hyaluronidases that may be used with this invention can be found in Table 3 (SEQ ID NO: 1-3).

[0092] Hyaluronidases include bacterial hyaluronidase (EC 4.2.2.1 or EC 4.2.99.1), hyaluronidases derived from leeches, other parasites, and crustaceans (EC 3.2.1.36), and mammalian hyaluronidases (EC 3.2.1.35). Hyaluronidases include, but are not limited to, those from rodents, dogs, cats, rabbits, birds, cattle, sheep, pigs, horses, fish, frogs, bacteria, and any hyaluronidase derived from leeches, other parasites, and crustaceans. Examples of hyaluronidases can be found, for example, in European Patent EP2833905B1, which is incorporated herein by reference in its entirety for its teachings concerning hyaluronidase. Types and classifications of hyaluronidases can be found in Jung H. Hyaluronidase: An overview of its properties, applications, and side effects. Arch Plast Surg. July 2020;47(4):297-300. doi: 10.5999 / aps.2020.00752, which is also incorporated in full by reference for its teachings on hyaluronidases. Specifically, HAases can be of mammalian origin (such as Homo sapiens or Bos taurus).

[0093] HAases can be naturally occurring, or, compared to naturally occurring or native HAases, can contain one or more mutations. HAase activity can be derived from a full-length protein or a truncated soluble protein fragment having HAase activity, or wherein the HAase contains one or more mutations compared to a native HAase molecule. For example, the HAases found in SEQ ID NO: 1-3 can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more mutations. This can represent 50%, 60%, 70%, 80%, 85%, 90%, or 95% (or any amount between, below, or above these percentages) of the native HAase. These mutations can be located in the amino acids of HAase or in the nucleic acids encoding HAase. These mutations can include, but are not limited to, expression systems encoding wild-type, point mutations, deletion mutations, insertion mutations, or codon-optimized sequences to produce the HAase-encoding DNA sequence (cds).

[0094] The HAase used with the methods and compositions disclosed herein can be naturally produced by cells that can be engineered to produce hyaluronidase. For example, the HAase can be derived from cells cultured in a culture medium, eliminating the need for exogenous addition of HAase. Cells that produce HAase can either naturally produce it or be engineered to produce it. When engineered, the cells can contain an expression system that synthesizes and / or contains recombinant nucleic acids encoding HAase.

[0095] Hyaluronidase can be used with various cell types and cell cultures, including adherent and suspension cultures. In adherent cell cultures, cells grow while attached to the substrate as a monolayer. In suspension cell cultures, cells float freely in the culture medium. An example of adherent cell cultures is the use of microcarriers. Microcarriers are well known in the art.

[0096] Microcarriers can be made from a variety of different materials. In some embodiments, microcarriers are made from non-biodegradable materials such as cellulose, DEAE-dextran, hydroxylated methacrylates, polyacrylamide, polystyrene, plastics, glass, ceramics, and silicone. In some embodiments, microcarriers are made from biodegradable materials such as collagen, alginate, dextran, gellan gum, and gelatin. These microcarrier materials, along with different surface chemicals, can influence cell behavior, including morphology and proliferation. Surface chemicals can include extracellular matrix proteins, recombinant proteins, peptides, and positively or negatively charged molecules. For example, microcarriers can be coated with collagen, fibronectin, pronectin, matrigel, laminin, hydatidin, or e-cadherin. Microcarriers can be positively charged or surface-modified using peptide conjugates (such as nanofibers) or thermally reversible / responsive surfaces (such as poly-N-isopropylacrylamide (poly-NIPAAM)). In some embodiments, microcarriers are magnetic beads.

[0097] Microcarriers preferably have a density that allows them to remain in suspension under gentle agitation. For example, microcarriers may have a specific gravity between 1.015 g / cc and 1.035 g / cc. Microcarrier cell culture is typically carried out in a rotary flask, but other containers, such as rotating-wall microgravity bioreactors or fluidized-bed bioreactors, can also support microcarrier-based cultures.

[0098] Several types of microcarriers are commercially available, including those based on alginate (GEM). TM Global Cell Solutions, based on dextran (Cytodex) TM GE Healthcare), based on collagen (Cultispher) TM (Percell) and polystyrene-based (SoloHill Engineering) microcarriers. They differ in porosity, specific gravity, optical properties, presence of animal components, and surface chemistry.

[0099] In this specific application, cells are cultured in bioreactors on microcarriers specifically for the production of EVs. During the growth phase, cells such as MSCs are first expanded on microcarriers, and the cultures are then used to produce EVs or other extracellular materials commonly used for therapeutic agent development. The culture medium for collecting EVs is typically (but not always) different from the culture medium used for cell growth. EVs are produced by cells cultured in an EV collection medium, and the longer the cells can be cultured, the more EVs are produced. During extended cultures of MSCs and other cells, the cell culture medium may become viscous in order to prolong the EV production phase, thus affecting the quality of the culture. This paper discloses that adding hyaluronidase to the culture medium allows for control of the medium viscosity, resulting in longer-lived cultures and higher productivity. It is also disclosed that hyaluronidase can be added to conditioned media after collection to aid purification, thereby enhancing yield. Many novel methods for improving cell culture media have been discovered and described herein for enhancing the production or purification of EVs and other cell-generated materials by adding hyaluronidase at optimal times, at optimal concentrations, and in optimal modes (single-dose versus repeated-dose).

[0100] Those skilled in the art will understand the cell types that can be used with the cell culture techniques described above. Cells used with the compositions disclosed herein can be any cell capable of being cultured and may include viable, live cells. Cells can be modified. For example, cells can be genetically modified to achieve permanent or temporary changes in the physicochemical characteristics, biodistribution, pharmacokinetics, pharmacodynamics, or biological function of the extracellular material.

[0101] In some embodiments, the disclosed compositions contain stem cells or progenitor cells. Examples of stem cells include pluripotent stem cells, adult stem cells, blastocyst-derived stem cells, germline-derived stem cells, teratoma-derived stem cells, totipotent stem cells, multipotent stem cells, tumor suppressor-independent stem cells (OISCs), embryonic stem cells (ES), embryonic germ cells (EG), and embryonic cancer cells (EC). Stem cells can possess a wide variety of different properties and categories of these properties. For example, in some forms, stem cells are capable of proliferating in an undifferentiated state to at least 10, 15, 20, 30, or more passages. In some forms, stem cells can proliferate for more than a year without differentiation. Stem cells can also maintain a normal karyotype during proliferation and / or differentiation. Stem cells can also maintain the ability to differentiate into mesodermal, endoderm, and ectoderm tissues (including germ cells, oocytes, and sperm). Some stem cells can also be cells capable of unlimited proliferation in vitro in an undifferentiated state. Some stem cells can also maintain a normal karyotype through long-term culture. Even after prolonged culture, some stem cells can maintain the potential to differentiate into derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm). Some stem cells can form any cell type in an organism. Some stem cells can form embryoids under certain conditions (such as growth on a culture medium that does not maintain undifferentiated growth). Some stem cells can form chimeras by fusing with, for example, blastocysts. Some stem cells can be induced or transformed from non-stem cells through genetic or chemical means.

[0102] In some embodiments, the disclosed compositions comprise cells other than stem cells. The adult human body produces many different cell types. These different cell types include, but are not limited to, keratinized epithelial cells, moist stratified barrier epithelial cells, exocrine epithelial cells, hormone-secreting cells, epithelial absorptive cells (intestine, exocrine glands, and urogenital tract), metabolic and storage cells, barrier function cells (lung, intestine, exocrine glands, and urogenital tract), epithelial cells located in the lining of closed internal body cavities, propulsive ciliated cells, extracellular matrix secretory cells, contractile cells, blood and immune system cells, sensory transduction cells, autonomic neurons, sensory organ and peripheral neuronal support cells, central nervous system neurons and glial cells, lens cells, pigment cells, germ cells, and caregiver cells.

[0103] Human cells include keratinized epithelial cells, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), keratinocytes of the nails and toenails, nail bed basal cells (stem cells), medullary hair axis cells, cortical hair axis cells, epidermal hair axis cells, epidermal root sheath cells, root sheath cells of Huxley's layer, root sheath cells of Henle's layer, outer root sheath cells, hair matrix cells (stem cells), moist stratified barrier epithelial cells, surface epithelial cells of the layered squamous epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina, basal cells of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina (stem cells), urothelial cells (located in the inner lining of the bladder and urethra), exocrine epithelial cells, salivary gland mucin cells (rich in polysaccharide secretions), salivary gland serous cells (rich in glycoprotein proteases secretions), and the von Willebrand gland in the tongue. Ebner's gland cells (wash taste buds), mammary gland cells (milk secretion), lacrimal gland cells (tear secretion), ceruminous gland cells in the ear (earwax secretion), dark cells of eccrine sweat glands (glycoprotein secretion), clear cells of eccrine sweat glands (small molecule secretion), apocrine sweat gland cells (odorous secretion, sensitive to sex hormones), Moll's gland cells in the eyelids (specialized sweat glands), sebaceous gland cells (lipid-rich sebum secretion), Bowman's gland cells in the nose (wash olfactory epithelium), Brunner's gland cells in the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secreting semen components, including fructose from motile sperm), prostate gland cells (secreting semen components), bulbourethral gland cells (mucus secretion), Bartholin's gland cells (vaginal lubricant secretion), and Little's gland cells. Littre cells (mucus secretion), endometrial cells (carbohydrate secretion), separate goblet cells of the respiratory and digestive tracts (mucus secretion), gastric parietal mucus cells (mucus secretion), gastric zymogen cells (pepsinogen secretion), gastric acid-secreting cells (HCl secretion), pancreatic acinar cells (bicarbonate and digestive enzyme secretion), Paneth cells of the small intestine (lysozyme secretion), type II lung cells of the lung (surfactant secretion), Clara cells of the lung(cells), hormone-secreting cells, anterior pituitary cells that secrete growth hormone, anterior pituitary cells that secrete follicle-stimulating hormone, anterior pituitary cells that secrete luteinizing hormone, anterior pituitary cells that secrete prolactin, anterior pituitary cells that secrete adrenocorticotropic hormone, anterior pituitary cells that secrete thyroid-stimulating hormone, middle pituitary cells that secrete melanocyte-stimulating hormone, posterior pituitary cells that secrete oxytocin, posterior pituitary cells that secrete vasopressin, intestinal and respiratory cells that secrete serotonin, intestinal and respiratory cells that secrete endorphins, intestinal and respiratory cells that secrete somatostatin, and intestinal and respiratory cells that secrete gastrin. Respiratory cells, intestinal and respiratory cells that secrete pancreatin, intestinal and respiratory cells that secrete cholecystokinin, intestinal and respiratory cells that secrete insulin, intestinal and respiratory cells that secrete glucagon, intestinal and respiratory cells that secrete leptin, thyroid cells that secrete thyroid hormones, thyroid cells that secrete calcitonin, parathyroid cells that secrete parathyroid hormone, parathyroid eosinophilic cells, adrenal cells that secrete adrenaline, adrenal cells that secrete norepinephrine, adrenal cells that secrete steroid hormones (mineralocorticoids and glucocorticoids), and interstitial cells of the testis that secrete testosterone (Leydig). Cells of the kidney, including: inner membrane cells of follicles secreting estrogen, luteal cells of ruptured follicles secreting progesterone, juxtaglomerular complex cells (renin secretion), dense macula cells of the kidney, peripolar cells of the kidney, mesangial cells of the kidney, epithelial absorptive cells (intestine, exocrine glands, and urogenital tract), brush border cells of the intestine (with microvilli), striate cells of exocrine glands, gallbladder epithelial cells, brush border cells of the proximal tubules of the kidney, cells of the distal tubules of the kidney, non-ciliated cells of the efferent tubules, chief cells of the epididymis, basal cells of the epididymis, metabolic and storage cells, hepatocytes (or liver cells), white adipocytes, brown adipocytes, hepatocytes, barrier function cells (in the lungs, intestines, exocrine glands, and urogenital tract), type I lung cells (located in the inner lining of the air sacs of the lungs), pancreatic duct cells (avesicular cells), non-striated ductal cells (of sweat glands, salivary glands, mammary glands, etc.), glomerular wall cells, glomerular foot cells, and loops of Henry.Henle cells (in the kidney), renal collecting duct cells, ductal cells (in the seminal vesicle, prostate, etc.), epithelial cells located in the inner lining of closed internal body cavities, fenestrated cells of vascular and lymphatic endothelium, continuous cells of vascular and lymphatic endothelium, spleen cells of vascular and lymphatic endothelium, synovial cells (located in the inner lining of joint cavities, secreting hyaluronic acid), serous cells (located in the inner lining of the peritoneum, pleura, and pericardial cavity), squamous cells (located in the inner lining of the perilymphatic space of the ear), squamous cells (located in the inner lining of the endolymphatic space of the ear), columnar cells of the endolymphatic space with microvilli (located in the inner lining of the endolymphatic space of the ear), columnar cells of the endolymphatic space without microvilli (located in the inner lining of the endolymphatic space of the ear), dark cells (located in the inner lining of the endolymphatic space of the ear), vestibular cells (located in the inner lining of the endolymphatic space of the ear), basal cells of the stria vascularis (located in the inner lining of the endolymphatic space of the ear), marginal cells of the stria vascularis (located in the inner lining of the endolymphatic space of the ear), Claudius cells (Cell of Claudius (located in the inner endolymphatic layer of the ear), Cellof Boettcher cells (located in the inner endolymphatic layer of the ear), choroid plexus cells (cerebrospinal fluid secretion), pia mater arachnoid squamous cells, pigmented ciliary epithelial cells of the eye, non-pigmented ciliary epithelial cells of the eye, corneal endothelial cells, propulsive ciliary cells, respiratory tract ciliary cells, fallopian tube ciliary cells (in females), endometrial ciliary cells (in females), reticulum testis ciliary cells (in males), efferent tubule ciliary cells (in males), ciliated ependymal cells of the central nervous system (located in the inner lining of the brain cavity), extracellular matrix secretory cells, ameloblastic epithelial cells (tooth enamel secretion), semilunar epithelial cells of the vestibular organ (proteoglycan secretion), and interdental epithelial cells of the organ of Corti. (Secretory covering the hair cells), loose connective tissue fibroblasts, corneal fibroblasts, tendon fibroblasts, bone marrow reticular tissue fibroblasts, other (non-epithelial) fibroblasts, capillary periepithelial cells, intervertebral disc nucleus pulposus cells, cementoblasts / cementocytes (secreting bone-like cementum from the tooth root), odontocytes / dentinocytes (secreting dentin), hyaline cartilage chondrocytes, fibrochondrocytes, elastic cartilage chondrocytes, osteoblasts / bone cells, osteoprogenitor cells (stem cells of osteoblasts), vitreous cells of the vitreous body, stellate cells of the extraaural lymphatic space, contractile cells. Cells, red skeletal muscle cells (slow), white skeletal muscle cells (fast), intermediate skeletal muscle cells, muscle spindle-nuclear cells, muscle spindle-nuclear chain cells, satellite cells (stem cells), ordinary cardiomyocytes, nodular cardiomyocytes, Purkinje fiber cells, smooth muscle cells (various types), myoepithelial cells of the iris, myoepithelial cells of exocrine glands, blood and immune system cells, erythrocytes (or red blood cells).Cells, megakaryocytes, monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglia (in the central nervous system), neutrophils, eosinophils, basophils, mast cells, helper T lymphocytes, suppressor T lymphocytes, cytotoxic T lymphocytes, IgM B lymphocytes, IgG B lymphocytes, IgA B lymphocytes, IgE cells. B lymphocytes, killer cells, stem cells and directed progenitor cells (various types) of the blood and immune system, sensory transducer cells, rod cells of photoreceptors in the eye, blue cone cells of photoreceptors in the eye, green cone cells of photoreceptors in the eye, red cone cells of photoreceptors in the eye, inner hair cells of the cochlea, outer hair cells of the cochlea, type I hair cells of the vestibular system (acceleration and gravity), type II hair cells of the vestibular system (acceleration and gravity), type I taste bud cells, olfactory neurons, basal cells of the olfactory epithelium (stem cells of olfactory neurons), type I carotid somatic cells (blood pH sensors), type II carotid somatic cells (blood pH sensors), Merkel cells of the epidermis. Touch sensor cells, tactile primary sensory neurons (various types), cold-sensitive primary sensory neurons, heat-sensitive primary sensory neurons, pain-sensitive primary sensory neurons (various types), proprioceptive primary sensory neurons (various types), autonomic neurons, cholinergic neurons (various types), adrenergic neurons (various types), peptidergic neurons (various types), supporting cells of sensory organs and peripheral neurons, inner column cells of the organ of Corti, outer column cells of the organ of Corti, inner finger cells of the organ of Corti, outer finger cells of the organ of Corti, boundary cells of the organ of Corti, Hensen cells of the organ of Corti, vestibular organ supporting cells, type I taste bud supporting cells, olfactory epithelial supporting cells, Schwann cells. Satellite cells (encapsulating peripheral nerve cell bodies), intestinal glial cells, central nervous system neurons and glial cells, neuronal cells (multiple types, classification still poor), astrocytes (various types), oligodendrocytes, lens cells, anterior lens epithelial cells, lens fiber cells containing lens proteins, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, spermatocytes, spermatogonia (spermocyte stem cells), nurse cells, follicular cells, Sertoli cells (in the testes), and thymic epithelial cells.

[0104] In some cases, the cells are mesenchymal stem cells (MSCs) or bone marrow stromal cells (BMSCs). These terms are used synonymously throughout this document. MSCs are of interest because they are readily isolated from small aspirates of bone marrow or other mesenchymal stem cell sources, and they readily generate single-cell-derived colonies. Bone marrow cells can be obtained from the iliac crest, femur, tibia, spine, ribs, knee, or other mesenchymal tissues. Other sources of MSCs include the embryonic yolk sac, placenta, umbilical cord, skin, fat, synovial tissue from joints, and blood. The presence of MSCs in cultured colonies can be verified by specific cell surface markers identified using monoclonal antibodies. See U.S. Patents 5,486,359 and 7,153,500. Single-cell-derived colonies can expand through up to 50 population doublings within approximately 10 weeks and can differentiate into osteoblasts, adipocytes, chondrocytes (Friedenstein et al., 1970 Cell Tissue Kinet. 3:393-403; Castro-Malaspina et al., 1980 Blood 56:289-301; Beresford et al., 1992 J. Cell Sci. 102:341-351; Prockop, 1997 Science 276:71-74), myocytes (Wakitani et al., 1995 MuscleNerve 18:1417-1426), astrocytes, oligodendrocytes, and neurons (Azizi et al., 1998 Proc. Natl. Acad. Sci. USA 95:3908-3913; Kopen et al., 1999 Proc. Natl. Acad. Sci. USA 96:10711-10716; Chopp et al., 2000 Neuroreport II 300 1-3005; Woodbury et al., 2000 Neuroscience Res. 61:364-370. In rare cases, cells can differentiate into all three lineages. Therefore, MSCs act as progenitor cells for a variety of mesenchymal cell lineages, including bone, cartilage, ligaments, tendons, fat, muscle, cardiac tissue, matrix, dermis, and other connective tissues. See U.S. Patents 6,387,369 and 7,101,704. For these reasons, the potential use of MSCs in cell and gene therapies for a variety of human diseases is currently being tested (Horwitz et al., 1999 Nat. Med. 5:309-313; Caplan et al., 2000 Clin. Orthoped. 379:567-570).

[0105] In some cases, MSCs can be defined by various biomarkers. For example, MSCs can be positive for CD73, CD90, and CD166, and negative for CD14, CD34, and CD45.

[0106] The cells can be derived from humans or other animals. For example, cells can be derived from mice, guinea pigs, rats, cattle, horses, pigs, sheep, or goats. In some implementations, the cells are derived from non-human primates. In some cases, the cells are used for autologous or allogeneic therapy.

[0107] Hyaluronidase can be applied to culture media in a wide range of amounts.

[0108] And has been found to be effective in amounts from 0.001 U / mL to 500 U / mL (see Example 1). Specifically, amounts contemplated herein are 0.001 U / mL, 0.002 U / mL, 0.003 U / mL, 0.004 U / mL, 0.005 U / mL, 0.006 U / mL, 0.007 U / mL, 0.008 U / mL, 0.009 U / mL, 0.01 U / mL, 0.02 U / mL, 0.03 U / mL, 0.04 U / mL, 0.05 U / mL, 0.06 U / mL, 0.07 U / mL, 0.08 U / mL, 0.09 U / mL, 0.10 U / mL, 0.20 U / mL, 0.30 U / mL, 0.40 U / mL, 0.50 U / mL, 0.60 U / mL, 0.70 U / mL, 0.80 U / mL, 0.90 U / mL, 1.0 U / mL, 1.1 U / mL, 1.2 U / mL, 1.3 U / mL, 1.4 U / mL, 1.5 U / mL, 1.6 U / mL, 1.7 U / mL, 1.8 U / mL, 1.9 U / mL, 2 U / mL, 2.1 U / mL, 2.2 U / mL, 2.3 U / mL, 2.4 U / mL, 2.5 U / mL, 2.6 U / mL, 2.7 U / mL, 2.8 U / mL, 2.9 U / mL, 3 U / mL, 3.1 U / mL, 3.2 U / mL, 3.3 U / mL, 3.4 U / mL, 3.5 U / mL, 3.6 U / mL, 3.7 U / mL, 3.8 U / mL, 3.9 U / mL, 4 U / mL, 4.1 U / mL, 4.2 U / mL, 4.3 U / mL, 4.4 U / mL, 4.5 U / mL, 4.6 U / mL, 4.7 U / mL, 4.8 U / mL, 4.9 U / mL, 5 U / mL, 5.1 U / mL, 5.2 U / mL, 5.3 U / mL, 5.4 U / mL, 5.5 U / mL, 5.6 U / mL, 5.7 U / mL, 5.8 U / mL, 5.9 U / mL, 6 U / mL, 6.1 U / mL, 6.2 U / mL, 6.3 U / mL, 6.4 U / mL, 6.5 U / mL, 6.6 U / mL, 6.7 U / mL, 6.8 U / mL, 6.9 U / mL, 7 U / mL, 7.1 U / mL, 7.2 U / mL, 7.3 U / mL, 7.4 U / mL, 7.5 U / mL, 7.6 U / mL, 7.7 U / mL, 7.8 U / mL, 7.9 U / mL, 8 U / mL, 8.1 U / mL, 8.2 U / mL, 8.3 U / mL, 8.4 U / mL, 8.5 U / mL, 8.6 U / mL, 8.7 U / mL, 8.8 U / mL, 8.9 U / mL, 9 U / mL, 9.1 U / mL, 9.2 U / mL, 9.3 U / mL, 9.4 U / mL, 9.5 U / mL, 9.6 U / mL, 9.7 U / mL, 9.8 U / mL, 9.9U / mL、10U / mL、10.1U / mL、10.2U / mL、10.3U / mL、10.4U / mL、10.5U / mL、10.6U / mL、10.7U / mL、10.8U / mL、10.9U / mL、11U / mL、11.1U / mL、11.2U / mL、11.3U / mL、11.4U / mL、11.5U / mL、11.6U / mL、11.7U / mL、11.8U / mL、11.9U / mL、12U / mL、12.1U / mL、12.2U / mL、12.3U / mL、12.4U / mL、12.5U / mL、12.6U / mL、12.7U / mL、12.8U / mL、12.9U / mL、13U / mL、13.1U / mL、13.2U / mL、13.3U / mL、13.4U / mL、13.5U / mL、13.6U / mL、13.7U / mL、13.8U / mL、13.9U / mL、14U / mL、14.1U / mL、14.2U / mL、14.3U / mL、14.4U / mL、14.5U / mL、14.6U / mL、14.7U / mL、14.8U / mL、14.9U / mL、15U / mL、15.1U / mL、15.2U / mL、15.3U / mL、15.4U / mL、15.5U / mL、15.6U / mL、15.7U / mL、15.8U / mL、15.9U / mL、16U / mL、16.1U / mL、16.2U / mL、16.3U / mL、16.4U / mL、16.5U / mL、16.6U / mL、16.7U / mL、16.8U / mL、16.9U / mL、17U / mL、17.1U / mL、17.2U / mL、17.3U / mL、17.4U / mL、17.5U / mL、17.6U / mL、17.7U / mL、17.8U / mL、17.9U / mL、18U / mL、18.1U / mL、18.2U / mL、18.3U / mL、18.4U / mL、18.5U / mL、18.6U / mL、18.7U / mL、18.8U / mL、18.9U / mL、19U / mL、19.1U / mL、19.2U / mL、19.3U / mL、19.4U / mL、19.5U / mL、19.6U / mL、19.7U / mL、19.8U / mL、19.9U / mL、20U / mL、20.1U / mL、20.2U / mL、20.3U / mL、20.4U / mL、20.5U / mL、20.6U / mL、20.7U / mL、20.8U / mL、20.9U / mL、21U / mL、21.1U / mL、21.2U / mL、21.3U / mL、21.4U / mL、21.5U / mL、21.6U / mL、21.7U / mL、21.8U / mL、21.9U / mL、22U / mL、22.1U / mL、22.2U / mL、22.3U / mL、22.4U / mL、22.5U / mL、22.6U / mL、22.7U / mL、22.8U / mL、22.9U / mL、23U / mL、23.1U / mL、23.2U / mL、23.3U / mL、23.4U / mL、23.5U / mL、23.6U / mL、23.7U / mL、23.8U / mL、23.9U / mL、24U / mL、24.1U / mL、24.2U / mL、24.3U / mL、24.4U / mL、24.5U / mL、24.6U / mL、24.7U / mL、24.8U / mL、24.9U / mL、25U / mL、25.1U / mL、25.2U / mL、25.3U / mL、25.4U / mL、25.5U / mL、25.6U / mL、25.7U / mL、25.8U / mL、25.9U / mL、26U / mL、26.1U / mL、26.2U / mL、26.3U / mL、26.4U / mL、26.5U / mL、26.6U / mL、26.7U / mL、26.8U / mL、26.9U / mL、27U / mL、27.1U / mL、27.2U / mL、27.3U / mL、27.4U / mL、27.5U / mL、27.6U / mL、27.7U / mL、27.8U / mL、27.9U / mL、28U / mL、28.1U / mL、28.2U / mL、28.3U / mL、28.4U / mL、28.5U / mL、28.6U / mL、28.7U / mL、28.8U / mL、28.9U / mL、29U / mL、29.1U / mL、29.2U / mL、29.3U / mL、29.4U / mL、29.5U / mL、29.6U / mL、29.7U / mL、29.8U / mL、29.9U / mL、30U / mL、30.1U / mL、30.2U / mL、30.3U / mL、30.4U / mL、30.5U / mL、30.6U / mL、30.7U / mL、30.8U / mL、30.9U / mL、31U / mL、31.1U / mL、31.2U / mL、31.3U / mL、31.4U / mL、31.5U / mL、31.6U / mL、31.7U / mL、31.8U / mL、31.9U / mL、32U / mL、32.1U / mL、32.2U / mL、32.3U / mL、32.4U / mL、32.5U / mL、32.6U / mL、32.7U / mL、32.8U / mL、32.9U / mL、33U / mL、33.1U / mL、33.2U / mL、33.3U / mL、33.4U / mL、33.5U / mL、33.6U / mL、33.7U / mL、33.8U / mL、33.9U / mL、34U / mL、34.1U / mL、34.2U / mL、34.3U / mL、34.4U / mL、34.5U / mL、34.6U / mL、34.7U / mL、34.8U / mL、34.9U / mL、35U / mL、35.1U / mL、35.2U / mL、35.3U / mL、35.4U / mL、35.5U / mL、35.6U / mL、35.7U / mL、35.8U / mL、35.9U / mL、36U / mL、36.1U / mL、36.2U / mL、36.3U / mL、36.4U / mL、36.5U / mL、36.6U / mL、36.7U / mL、36.8U / mL、36.9U / mL、37U / mL、37.1U / mL、37.2U / mL、37.3U / mL、37.4U / mL、37.5U / mL、37.6U / mL、37.7U / mL、37.8U / mL、37.9U / mL、38U / mL、38.1U / mL、38.2U / mL、38.3U / mL、38.4U / mL、38.5U / mL、38.6U / mL、38.7U / mL、38.8U / mL、38.9U / mL、39U / mL、39.1U / mL、39.2U / mL、39.3U / mL、39.4U / mL、39.5U / mL、39.6U / mL、39.7U / mL、39.8U / mL、39.9U / mL、40U / mL、40.1U / mL、40.2U / mL、40.3U / mL、40.4U / mL、40.5U / mL、40.6U / mL、40.7U / mL、40.8U / mL、40.9U / mL、41U / mL、41.1U / mL、41.2U / mL、41.3U / mL、41.4U / mL、41.5U / mL、41.6U / mL、41.7U / mL、41.8U / mL、41.9U / mL、42U / mL、42.1U / mL、42.2U / mL、42.3U / mL、42.4U / mL、42.5U / mL、42.6U / mL、42.7U / mL、42.8U / mL、42.9U / mL、43U / mL、43.1U / mL、43.2U / mL、43.3U / mL、43.4U / mL、43.5U / mL、43.6U / mL、43.7U / mL、43.8U / mL、43.9U / mL、44U / mL、44.1U / mL、44.2U / mL、44.3U / mL、44.4U / mL、44.5U / mL、44.6U / mL、44.7U / mL、44.8U / mL、44.9U / mL、45U / mL、45.1U / mL、45.2U / mL、45.3U / mL、45.4U / mL、45.5U / mL、45.6U / mL、45.7U / mL、45.8U / mL、45.9U / mL、46U / mL、46.1U / mL、46.2U / mL、46.3U / mL、46.4U / mL、46.5U / mL、46.6U / mL、46.7U / mL、46.8U / mL、46.9U / mL、47U / mL、47.1U / mL、47.2U / mL、47.3U / mL、47.4U / mL、47.5U / mL、47.6U / mL、47.7U / mL、47.8U / mL、47.9U / mL、48U / mL、48.1U / mL、48.2U / mL、48.3U / mL、48.4U / mL、48.5U / mL、48.6U / mL、48.7U / mL、48.8U / mL、48.9U / mL、49U / mL、49.1U / mL、49.2U / mL、49.3U / mL、49.4U / mL、49.5U / mL、49.6U / mL、49.7U / mL、49.8U / mL、49.9U / mL、50U / mL、50.1U / mL、50.2U / mL、50.3U / mL、50.4U / mL、50.5U / mL、50.6U / mL、50.7U / mL、50.8U / mL、50.9U / mL、51U / mL、51.1U / mL、51.2U / mL、51.3U / mL、51.4U / mL、51.5U / mL、51.6U / mL、51.7U / mL、51.8U / mL、51.9U / mL、52U / mL、52.1U / mL、52.2U / mL、52.3U / mL、52.4U / mL、52.5U / mL、52.6U / mL、52.7U / mL、52.8U / mL、52.9U / mL、53U / mL、53.1U / mL、53.2U / mL、53.3U / mL、53.4U / mL、53.5U / mL、53.6U / mL、53.7U / mL、53.8U / mL、53.9U / mL、54U / mL、54.1U / mL、54.2U / mL、54.3U / mL、54.4U / mL、54.5U / mL、54.6U / mL、54.7U / mL、54.8U / mL、54.9U / mL、55U / mL、55.1U / mL、55.2U / mL、55.3U / mL、55.4U / mL、55.5U / mL、55.6U / mL、55.7U / mL、55.8U / mL、55.9U / mL、56U / mL、56.1U / mL、56.2U / mL、56.3U / mL、56.4U / mL、56.5U / mL、56.6U / mL、56.7U / mL、56.8U / mL、56.9U / mL、57U / mL、57.1U / mL、57.2U / mL、57.3U / mL、57.4U / mL、57.5U / mL、57.6U / mL、57.7U / mL、57.8U / mL、57.9U / mL、58U / mL、58.1U / mL、58.2U / mL、58.3U / mL、58.4U / mL、58.5U / mL、58.6U / mL、58.7U / mL、58.8U / mL、58.9U / mL、59U / mL、59.1U / mL、59.2U / mL、59.3U / mL、59.4U / mL、59.5U / mL、59.6U / mL、59.7U / mL、59.8U / mL、59.9U / mL、60U / mL、60.1U / mL、60.2U / mL、60.3U / mL、60.4U / mL、60.5U / mL、60.6U / mL、60.7U / mL、60.8U / mL、60.9U / mL、61U / mL、61.1U / mL、61.2U / mL、61.3U / mL、61.4U / mL、61.5U / mL、61.6U / mL、61.7U / mL、61.8U / mL、61.9U / mL、62U / mL、62.1U / mL、62.2U / mL、62.3U / mL、62.4U / mL、62.5U / mL、62.6U / mL、62.7U / mL、62.8U / mL、62.9U / mL、63U / mL、63.1U / mL、63.2U / mL、63.3U / mL、63.4U / mL、63.5U / mL、63.6U / mL、63.7U / mL、63.8U / mL、63.9U / mL、64U / mL、64.1U / mL、64.2U / mL、64.3U / mL、64.4U / mL、64.5U / mL、64.6U / mL、64.7U / mL、64.8U / mL、64.9U / mL、65U / mL、65.1U / mL、65.2U / mL、65.3U / mL、65.4U / mL、65.5U / mL、65.6U / mL、65.7U / mL、65.8U / mL、65.9U / mL、66U / mL、66.1U / mL、66.2U / mL、66.3U / mL、66.4U / mL、66.5U / mL、66.6U / mL、66.7U / mL、66.8U / mL、66.9U / mL、67U / mL、67.1U / mL、67.2U / mL、67.3U / mL、67.4U / mL、67.5U / mL、67.6U / mL、67.7U / mL、67.8U / mL、67.9U / mL、68U / mL、68.1U / mL、68.2U / mL、68.3U / mL、68.4U / mL、68.5U / mL、68.6U / mL、68.7U / mL、68.8U / mL、68.9U / mL、69U / mL、69.1U / mL、69.2U / mL、69.3U / mL、69.4U / mL、69.5U / mL、69.6U / mL、69.7U / mL、69.8U / mL、69.9U / mL、70U / mL、70.1U / mL、70.2U / mL、70.3U / mL、70.4U / mL、70.5U / mL、70.6U / mL、70.7U / mL、70.8U / mL、70.9U / mL、71U / mL、71.1U / mL、71.2U / mL、71.3U / mL、71.4U / mL、71.5U / mL、71.6U / mL、71.7U / mL、71.8U / mL、71.9U / mL、72U / mL、72.1U / mL、72.2U / mL、72.3U / mL、72.4U / mL、72.5U / mL、72.6U / mL、72.7U / mL、72.8U / mL、72.9U / mL、73U / mL、73.1U / mL、73.2U / mL、73.3U / mL、73.4U / mL、73.5U / mL、73.6U / mL、73.7U / mL、73.8U / mL、73.9U / mL、74U / mL、74.1U / mL、74.2U / mL、74.3U / mL、74.4U / mL、74.5U / mL、74.6U / mL、74.7U / mL、74.8U / mL、74.9U / mL、75U / mL、75.1U / mL、75.2U / mL、75.3U / mL、75.4U / mL、75.5U / mL、75.6U / mL、75.7U / mL、75.8U / mL、75.9U / mL、76U / mL、76.1U / mL、76.2U / mL、76.3U / mL、76.4U / mL、76.5U / mL、76.6U / mL、76.7U / mL、76.8U / mL、76.9U / mL、77U / mL、77.1U / mL、77.2U / mL、77.3U / mL、77.4U / mL、77.5U / mL、77.6U / mL、77.7U / mL、77.8U / mL、77.9U / mL、78U / mL、78.1U / mL、78.2U / mL、78.3U / mL、78.4U / mL、78.5U / mL、78.6U / mL、78.7U / mL、78.8U / mL、78.9U / mL、79U / mL、79.1U / mL、79.2U / mL、79.3U / mL、79.4U / mL、79.5U / mL、79.6U / mL、79.7U / mL、79.8U / mL、79.9U / mL、80U / mL、80.1U / mL、80.2U / mL、80.3U / mL、80.4U / mL、80.5U / mL、80.6U / mL、80.7U / mL、80.8U / mL、80.9U / mL、81U / mL、81.1U / mL、81.2U / mL、81.3U / mL、81.4U / mL、81.5U / mL、81.6U / mL、81.7U / mL、81.8U / mL、81.9U / mL、82U / mL、82.1U / mL、82.2U / mL、82.3U / mL、82.4U / mL、82.5U / mL、82.6U / mL、82.7U / mL、82.8U / mL、82.9U / mL、83U / mL、83.1U / mL、83.2U / mL、83.3U / mL、83.4U / mL、83.5U / mL、83.6U / mL、83.7U / mL、83.8U / mL、83.9U / mL、84U / mL、84.1U / mL、84.2U / mL、84.3U / mL、84.4U / mL、84.5U / mL、84.6U / mL、84.7U / mL、84.8U / mL、84.9U / mL、85U / mL、85.1U / mL、85.2U / mL、85.3U / mL、85.4U / mL、85.5U / mL、85.6U / mL、85.7U / mL、85.8U / mL、85.9U / mL、86U / mL、86.1U / mL、86.2U / mL、86.3U / mL、86.4U / mL、86.5U / mL、86.6U / mL、86.7U / mL、86.8U / mL、86.9U / mL、87U / mL、87.1U / mL、87.2U / mL、87.3U / mL、87.4U / mL、87.5U / mL、87.6U / mL、87.7U / mL、87.8U / mL、87.9U / mL、88U / mL、88.1U / mL、88.2U / mL、88.3U / mL、88.4U / mL、88.5U / mL、88.6U / mL、88.7U / mL、88.8U / mL、88.9U / mL、89U / mL、89.1U / mL、89.2U / mL、89.3U / mL、89.4U / mL、89.5U / mL、89.6U / mL、89.7U / mL、89.8U / mL、89.9U / mL、90U / mL、90.1U / mL、90.2U / mL、90.3U / mL、90.4U / mL、90.5U / mL、90.6U / mL、90.7U / mL、90.8U / mL、90.9U / mL、91U / mL、91.1U / mL、91.2U / mL、91.3U / mL、91.4U / mL、91.5U / mL、91.6U / mL、91.7U / mL、91.8U / mL、91.9U / mL、92U / mL、92.1U / mL、92.2U / mL、92.3U / mL、92.4U / mL、92.5U / mL、92.6U / mL、92.7U / mL、92.8U / mL、92.9U / mL、93U / mL、93.1U / mL、93.2U / mL、93.3U / mL、93.4U / mL、93.5U / mL、93.6U / mL、93.7U / mL、93.8U / mL、93.9U / mL、94U / mL、94.1U / mL、94.2U / mL、94.3U / mL、94.4U / mL、94.5U / mL、94.6U / mL、94.7U / mL、94.8U / mL、94.9U / mL、95U / mL、95.1U / mL、95.2U / mL、95.3U / mL、95.4U / mL、95.5U / mL、95.6U / mL、95.7U / mL、95.8U / mL、95.9U / mL、96U / mL、96.1U / mL、96.2U / mL、96.3U / mL、96.4U / mL、96.5U / mL、96.6U / mL、96.7U / mL、96.8U / mL、96.9U / mL、97U / mL、97.1U / mL、97.2U / mL、97.3U / mL、97.4U / mL、97.5U / mL、97.6U / mL、97.7U / mL、97.8U / 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mL、494.2U / mL、494.3U / mL、494.4U / mL、494.5U / mL、494.6U / mL、494.7U / mL、494.8U / mL、494.9U / mL、495U / mL、495.1U / mL、495.2U / mL、495.3U / mL、495.4U / mL、495.5U / mL、495.6U / mL、495.7U / mL、495.8U / mL、495.9U / mL、496U / mL、496.1U / mL、496.2U / mL、496.3U / mL、496.4U / mL、496.5U / mL、496.6U / mL、496.7U / mL、496.8U / mL、496.9U / mL、497U / mL、497.1U / mL、497.2U / mL、497.3U / mL、497.4U / mL、497.5U / mL、497.6U / mL、497.7U / mL、497.8U / mL、497.Hyaluronidase may be used in cell cultures at concentrations of 9 U / mL, 498 U / mL, 498.1 U / mL, 498.2 U / mL, 498.3 U / mL, 498.4 U / mL, 498.5 U / mL, 498.6 U / mL, 498.7 U / mL, 498.8 U / mL, 498.9 U / mL, 499 U / mL, 499.1 U / mL, 499.2 U / mL, 499.3 U / mL, 499.4 U / mL, 499.5 U / mL, 499.6 U / mL, 499.7 U / mL, 499.8 U / mL, 499.9 U / mL, or 500 U / mL, or at any ratio of concentrations higher, lower, or between these values.

[0109] In a specific example, hyaluronidase is applied in the range of 0.001 U / ml to 10 U / ml. Those skilled in the art can readily assess the rate of hyaluronidase application based on the type of cell culture used, the specific viscosity issues faced, and the desired viscosity of the culture. For example, when MSCs are cultured to densities between 100,000 cells / mL and 1,000,000 cells / mL, using hyaluronidase at 0.001 U / ml (or 1 mU / ml) may be optimal. As MSCs reach higher densities more quickly, viscosity accumulates more rapidly, likely due to cells releasing hyaluronic acid secretions into the culture medium. Over time, HA accumulation leads to increased medium viscosity, eventually resulting in culture failure. Adding hyaluronidase breaks down HA and reduces viscosity to a level that allows the microcarriers to remain suspended, and the culture produces more EVs. If higher concentrations of hyaluronidase are added (e.g., 0.1 U / mL or higher), cells may detach from the microcarriers and cease growth.

[0110] Once MSCs generate EVs in small-scale flasks or large-scale bioreactors, the culture medium is processed to purify the secretome containing various extracellular materials, including EVs. Conditioned medium must be clarified by deep filtration before TFF and chromatography. This clarification step can begin with a large cutoff filter of several micrometers, followed by smaller filters (e.g., 0.8 micrometers, and then 0.45 micrometers). This typically begins by removing all large (approximately 250 micrometers in diameter) microcarriers from the conditioned medium using a 60–100 micrometer sieve. Remaining cells and cell debris are usually removed with a 5-micrometer filter, and the medium is further clarified before TFF using 0.8-micrometer or 0.45-micrometer filters. A significant challenge within EVs is that even filters as large as 5 micrometers can rapidly clog the conditioned medium, and back pressure can create as biomaterial accumulates in the filter, causing many EVs to become irreversibly immobilized, significantly reducing yield. Ti has found that adding hyaluronidase to conditioned medium streamlines the filtration process, reduces stress buildup, and, surprisingly, significantly increases the amount of EV passing through the filter. This article presents several examples of increased yields for filters ranging in size from 0.45 microns to 5 microns.

[0111] During culture, hyaluronidase can be applied to the cell culture once or multiple times during the culture period, depending on the rate of viscosity buildup. For example, hyaluronidase can be applied when the cell culture is established or just before cell harvesting. It can be applied once daily, twice daily, three times daily, four times daily, or more than once daily. It can be applied every other day, or every three days, or every four days, or every five days, or every six days, or once weekly, twice weekly, three times weekly, four times weekly, five times weekly, or six times weekly. For example, it can be applied every week, every three weeks, every four weeks, or monthly. It can be applied at prescribed intervals or occasionally as needed. For example, those skilled in the art can assess when it should be applied and apply it at that time. Examples of timing are given in Example 1. Specific examples include applying hyaluronidase at least once between day 1 and day 30, or between day 5 and day 15, or between day 8 and day 12. Within this period, it can be applied once, twice, three times, four times, five times, or more.

[0112] Hyaluronidase can be added to cell cultures at various temperatures. For example, this paper discloses the addition of hyaluronidase to cell cultures at temperatures of 0℃, 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, 10℃, 11℃, 12℃, 13℃, 14℃, 15℃, 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, and 37℃. Hyaluronidase may be added at the following temperatures: 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, or 65℃, or at any temperature above, below, or in between.

[0113] Hyaluronidase can be added to cell cultures during conditions designed to improve the yield of desired products from cell cultures. These jointly optimized conditions include, for example, different agitation rates and culture medium compositions, where the addition can reduce the viscosity and / or contamination of cell culture materials and loss of cell suspension. “Variable agitation” means that the agitation rate can range from 20 RPM to 500 RPM, or a rate sufficient to maintain cell and / or microcarrier suspension without interfering with cell adhesion to microcarriers or cell viability. “Culture medium composition” means that cells (such as MSCs) can be grown in a variety of culture medium systems ranging from DMEM + FBS, DMEM + hPL, or αMEM + FBS or hPL. Serum-free media are also considered. It is entirely reasonable to expect that when any of these media are used for extended culture of EV-generating cells (such as MSCs), hyaluronidase can help maintain a healthy suspension culture or help filter and purify TV from these media.

[0114] Providing cell cultures with hyaluronidase can significantly reduce the viscosity of said cell cultures. For example, in application, the viscosity can be reduced by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, and 46%. 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any amount below or between these values. Compare this value to a cell culture control, where conditions are kept identical except that hyaluronidase was not added to the control.

[0115] This article discloses a cell culture medium containing hyaluronidase. This cell culture medium can be specific to, for example, MSCs, and can include cells as described above. The cell culture medium can be RoosterCollect EV Pro. TM RoosterCollect TM -EV or EV Boost TM Cell culture media may contain microcarriers or may be concentrated, as described above. Hyaluronidase may be provided in various concentrations, as described above.

[0116] This article also discloses a method for increasing the yield of extracellular vesicles (EVs) during downstream processing. The conditioned medium produced in the upstream process is a complex mixture containing not only extracellular vesicles but also other biological components, including cell debris, proteins, nucleic acids, lipids, and culture medium components. Large-scale biomanufacturing of EVs for clinical applications involves a downstream process that concentrates, purifies, and formulates EV formulations to meet final product specifications without compromising their critical quality attributes (CQAs).

[0117] Products obtained from the methods and compositions disclosed herein can be used in a variety of ways. These include, but are not limited to, therapeutic, veterinary, diagnostic, human health, cosmetic, and / or therapeutic diagnostic uses.

[0118] Specifically, this paper also discloses a method for improving the properties of a liquid cell culture medium, the method comprising culturing cells in the liquid culture medium, removing the cells, and then introducing HAase into the liquid culture medium after cell removal. By doing so, the viscosity of the culture medium can be significantly reduced. The reduction in viscosity has been discussed above. The reduction in viscosity can lead to an increase in yield. The increase in yield compared to a control without hyaluronidase can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%. An increase of 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or an increase of 2, 3, 4, 5, 6, 7, 8, 9 or 10 times or more in yield.

[0119] HAase can improve the yield, quality, or processing efficiency of one or more products secreted by cells, including extracellular vesicles (EVs), exosomes, microvesicles, cellular ghosts, secretory microparticles, or viral particles, as discussed herein. The method of using HAase during downstream processing is described in Example 3 below. Cells can be removed from liquid culture medium (or liquid culture medium can be removed from cells), and HAase can then be added. Once this occurs, the liquid culture medium can be clarified and concentrated. Such clarification can be performed by filtration. The filter can have a pore size, for example, between 0.45 µm and 100 µm. Then, the liquid culture medium can be concentrated by 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, and 53 times. 54 times, 55 times, 56 times, 57 times, 58 times, 59 times, 60 times, 61 times, 62 times, 63 times, 64 times, 65 times, 66 times, 67 times, 68 times, 69 times, 70 times, 71 times, 72 times, 73 times, 74 times, 75 times, 76 times, 77 times, 78 times, 79 times, 80 times, 81 times, 82 times, 83 times, 84 times, 85 times, 86 times, 87 times, 88 times, 89 times, 90 times, 91 times, 92 times, 93 times, 94 times, 95 times, 96 times, 97 times, 98 times, 99 times, or 100 times, or any amount lower than these values, higher than these values, or in between these values. The amount of HAase that can be added has been discussed above and in Example 3.

[0120] Specifically, this paper discloses a method comprising: generating extracellular vesicles in a bioreactor; and introducing hyaluronidase into the extracellular vesicles during downstream processing, thereby increasing the yield of extracellular vesicles compared to a control in which EVs are not exposed to hyaluronidase.

[0121] The addition of hyaluronidase during downstream processing can occur at any point before filtration. For example, it can be added directly to the bioreactor before collection, or it can be added after cells and microcarriers have been removed from the culture medium. It can be added before clarification, as described above. Hyaluronidase improves filtration and purification at 37°C, at room temperature, and even at 4°C. It can be added before or during the clarification step. It can be added at more than one of these time points, for example, at various time points during downstream processing.

[0122] This document further discloses a composition comprising a liquid cell culture medium and exogenously added hyaluronidase. "Exogenously added" means that the HAase is not naturally produced by a single cell within the culture, or is not produced in sufficient quantity to have the desired properties, but is added separately to the liquid cell culture medium. This liquid cell culture medium may or may not contain cells, such as MSCs. In some embodiments, cells are attached to microcarriers located within the liquid cell culture medium.

[0123] In one specific embodiment, the cells in the composition may be at a density of 50,000 cells / mL to 5,000,000 cells / mL, the EVs may be at a concentration of 1E8 particles / mL to 1E11 particles / mL, and the exogenous hyaluronidase may be present at a concentration between 0.0001 U / mL and 100 U / mL. The liquid cell culture medium of the composition may contain one or more products secreted by the cells, including extracellular vesicles (EVs), exosomes, microvesicles, ghost cells, secretory microparticles, or viral particles.

[0124] This document also discloses a composition comprising a conditioned medium derived from MSC cell cultures, wherein the conditioned medium contains EVs, wherein the EVs are present in a concentration between 1E8 and 1E11, and further wherein the conditioned medium contains exogenously added HAase at a concentration of about 0.01 U / mL to 100 U / mL, wherein the conditioned medium has not undergone clarification or filtration.

[0125] A kit is also disclosed comprising hyaluronidase and cell culture medium. The hyaluronidase may be provided separately from the cell culture medium or may be contained within the cell culture medium. The kit may further include instructions for use. These instructions may include specific details regarding the amount of hyaluronidase, application time, or concentration used. Other components may also be provided within the kit. The hyaluronidase may be provided in the amounts specified above. The cell culture medium may be of any type disclosed herein and may be concentrated or may contain microcarriers.

[0126] Example

[0127] Example 1: Composition and method for minimizing the viscosity of culture supernatant from primary cells.

[0128] MSC cell culture was performed using a standardized expansion process, although there were some variations in EV production, which allowed for the creation of conditioned media to purify the EVs. MSCs used in these studies were commercially available from RoosterBio Inc. as the RoosterVial product, derived from human bone marrow, human umbilical cord, or human adipose tissue. RoosterVial cells were thawed and cultured in Corning Cell Bind flasks at a concentration of 2000-3000 cells / cm³. 2Plate the cells and amplify them in Rooster Nourish-XF (a heterologous bioprocessing amplification medium) for 4–5 days or until 80–90% confluence. Alternative supplier media may be used and will be called upon. After a single passage, collect cells using TrypLE (Thermo) and passage them into Corning Cell Bind flasks, or seed them at 23,000 cells / mL into a bioreactor (disposable Corning rotary flasks or Sartorius Ambr 250 mL containers) and amplify them on microcarriers (Corning Synthemax or Sartorius Solohill collagen-coated carriers) supplemented with 42 mg / mL. These methods have been published and can be found in the following literature: Lembong, J., Kirian, R., Takacs, JD, Olsen, TR, Lock, LT, Rowley, JA, Ahsan, T. (2020) Bioreactor Parameters for Microcarrier-Based Human MSC Expansion under Xeno-Free Conditions in a Vertical-Wheel System, Bioengineering, 7, 73; and Lenzini, S., Jung, J., Patel, G., Jadhav, S., Gupta, P., Ladi, R., Boychyn, M., Rowley, JA, Zakhem, E. (2023) “Successful Development of a Scalable and Robust Process for MSC-EV Production” at the International Society for Cell & Gene Therapy Annual Meeting, Paris, France. Cells were expanded in RoosterNourish medium for 5 days, with RoosterReplenish added on day 3. Five days later, remove the Rooster-Nourish amplification medium, gently wash the cells off the microcarriers, and then add EV collection medium, typically for five days of collection. Alternatively, a single medium called HD-EV can be added on day 0 at the same concentration of 23,000 cells / ml and 42 mg / mL microcarriers, fed throughout the cycle without changing the medium.HD-EV is a chemically defined culture medium that enables up to 12 days of culture amplification and EV collection (T. Willstaedt, A. Walde, J. Rowley (2024) A Fed-Batch Chemically Defined hMSC-EV Bioprocess Medium Enabling 2-4x EV Yield Improvements in Bioreactor Culture at the International Society for Cell & Gene Therapy Annual Meeting, Vancouver, Canada).

[0129] The first experiment demonstrated the challenge of expanding MSCs in a 3D bioreactor using RoosterNourish. During bioreactor culture, the EV collection medium was either RoosterCollect-EV (RoosterBio, designated Collection Medium A) or RoosterCollect-EV-Pro (designated Collection Medium B). Collection Medium B is a richer medium designed to promote cell growth during EV collection, resulting in higher EV production levels (Reference: Trempl, M., Adlerz, K., Rowley, JA (2021) “Enhancing hMSC Extracellular Vesicle Productivity with a Novel Collection Media for Scalable MSC-EV Generation” at the International Society for Cell & Gene Therapy Annual Meeting, New Orleans). Figure 1B shows the cell culture suspension maintained up to day 10 in Medium A. In Medium B, the cell culture became highly viscous by day 5, and cells and microcarriers detached from the suspension. Adding dextran sulfate (a common cell culture additive used to address cell aggregation issues) to medium B in an attempt to resolve culture failure was ineffective. The observed increase in the viscosity of medium B over time led to greater aggregation of microcarriers, eventually increasing in weight to the point where they could no longer maintain suspension. As the aggregates became too large, the inner cells could not access the necessary nutrients, and the EVs secreted by the inner cells might not reach the culture medium solution to be collected. This is illustrated in Figure 1. The viscosity of the medium during a typical 10-day bioreactor cell culture, illustrating the phenomenon we were addressing, was quantified using internal viscosity measurements and calculations, as shown in Figure 1. Figure 2 As shown, the report is a viscosity index. In short, the viscosity of the cell culture medium is measured and recorded as a viscosity index, or [(viscosity-time)]. 样品 (Viscosity time) 水对照 )] / (viscosity time 水对照 Viscosity time refers to the total migration time of 1 mL of liquid solution from the syringe attached to the ring support through a standard 21-gauge blunt needle. The viscosity index is controlled to account for different relative humidity and atmospheric conditions.

[0130] Cells appear to begin secreting high-molecular-weight extracellular matrix (EM) molecules into the culture medium, which increases liquid viscosity even at low concentrations. These can include collagen, proteoglycans, and the like. Novel cell culture additive solutions containing hyaluronidase (HYAL) have now been shown to reduce and help maintain low medium viscosity during culture, enabling extended cell cultures in microcarrier bioreactors at appropriate times and concentrations without failure.

[0131] To test this, several types of enzymes were added to the viscous medium from MSC bioreactor cultures on day 10 (Figure 3). Preliminary tests were conducted. Figure 3A The preliminary test compared the effects of collagenase and Accumax. TM TyrpLE TM The effect of adding hyaluronidase (isolated from bovine testis, Sigma Alderich) to cells in culture on viscosity was investigated. The results clearly demonstrate that hyaluronidase has a significant effect on viscosity by significantly reducing it. A significant difference in viscosity was only observed with the addition of hyaluronidase compared to the untreated control. Figure 3A To further refine these results, TrypLE and trypsin were used as controls, and different amounts of hyaluronidase were added. Figure 3B Hyaluronidase at concentrations ranging from 100 U / mL to 10 mU / mL reduced the viscosity of the resulting culture medium, but 0.1 mU / mL was insufficient to affect the viscosity. Figure 3B TrypplLE and trypsin did not significantly reduce the viscosity of the culture medium. EV particles were tested under all conditions. Figure 3C Furthermore, particle counts (by NTA counting) were consistent under all conditions, indicating that enzymatic treatment did not destroy lipid nanoparticles as expected.

[0132] Another experiment was conducted ( Figure 4 The recombinant hyaluronidase (rPH20, ACRO Biosystems) at three concentrations (10 mU / mL, 1.0 mU / mL, and 0.1 mU / mL) was compared with bovine hyaluronidase at 1.0 mU / mL. Viscosity indexes were calculated on days 5, 7, 10, and 12 in five identical bioreactor cultures. Hyaluronidase was added on day 7. On days 10 and 12, the viscosity of the untreated culture climbed to 1.5 or higher, while the viscosity under the treated conditions decreased in a dose-dependent manner, with bovine hyaluronidase and recombinant human hyaluronidase showing equivalent viscosities at 1.0 mU / mL. This demonstration is significant because bovine material is unsuitable for pharmaceutical applications, and recombinant human material can be sourced at GMP quality.

[0133] Next, the toxicity of hyaluronidase to MSCs in culture was determined. This was a 2D test using MSFv03 medium, a commercially available test version of HD-EV (RoosterBio) medium (a chemically defined medium designed for cell expansion and EV collection). Hyaluronidase was added to cells in passage 3 on day 4 of culture, and cell health was monitored by morphology (images at 10x magnification) for 3 days. Waste medium was analyzed for p / ml and viscosity using a syringe method. Hyaluronidase was tested at concentrations of 0 U / ml, 10 U / ml, 30 U / ml, 100 U / ml, 300 U / ml, and 500 U / ml. Even at concentrations up to 500 U / ml, hyaluronidase had no negative impact on MSC morphology / viability during flask culture, and the cells remained adherent to the culture surface and were healthy.

[0134] This experiment demonstrated that adding up to 500 U / mL of HYAL had no negative impact on the morphology and overall health of BM-MSC cells in 2D cell cultures. On day 3, the dose-response of hyaluronidase at 10 U / mL, 100 U / mL, or 250 U / mL was tested in both flask and bioreactor cultures. Figure 10A The equivalent cell morphology and growth under all conditions in the flask are shown. Figure 10B Equivalent cell counts are shown, and 10C indicates equivalent viability of cells grown in flasks. However, when HYAL was added to 3D cultures on microcarriers in HD-EV medium, all hyaluronidase concentrations negatively impacted cell attachment to the microcarriers. Even with a low concentration of 10 U / mL added on day 3, cells detached from the microcarriers, and cell growth was impaired, as can be seen from the lack of staining in the cells. Culture was terminated after day 7.

[0135] In the final dose discovery experiment, MSC microcarrier cultures in HD-EV medium were administered at the following doses: 100 U / mL, 1.0 U / mL, 10 mU / mL, and 0.1 mU / mL. Figure 8 Viscosity measurements showed that the lowest dose of 0.1 mU / mL did not reduce viscosity, but doses of 10 mU / mL and higher did. Figure 12A Final dose studies of 1.0 mU / mL, 0.1 mU / mL, and 0.01 mU / mL are shown, with macroscopic failures at 0.1 mU / mL and 0.01 mU / mL observed. Figure 12B As shown in the diagram. Therefore, 1 mU / mL or 0.001 U / mL is the recommended working concentration for standard MSC culture under the previously described conditions. Figures 13A-CIt was demonstrated that 1 mU can maintain low culture viscosity, with optimal particle production (Fig. 13B) and particle size profiles being consistent (Fig. 13C). It is possible that cells with higher concentrations leading to faster hyaluronic acid production may require even higher concentrations, but it is important to maintain a balance between maintaining low culture viscosity and good cell adhesion to the microcarrier.

[0136] These data represent a new use case for hyaluronidase in cell cultures. MSC culture viscosity is not a major issue for MSCs, as these cells have not yet been used as production cells to manufacture bioproducts such as EVs. Therefore, as bioengineers strive to maximize cell culture yields and productivity to optimize the cost curve for these expensive products, there is a need to extend the culture life of MSC bioreactor cultures. Adding hyaluronidase in these micro-dose doses allows cell growth and EV production to proceed for longer periods than without hyaluronidase treatment, and typical methods for reducing culture contamination (such as dextran sulfate or PEG) do not address this challenge.

[0137] On the day of MSC-EV collection in a 15L bioreactor (5 days of amplification in RoosterNourish / Replenish, followed by 5 days of EV collection after medium exchange to RoosterCollect-EV on day 5), the collected material was significantly more viscous than the material produced in previous small-scale Ambr studies or even the 250 mL Ambr control in the same experiment. Treatment of the viscous medium with 10 U / mL hyaluronidase prior to processing significantly reduced the viscosity of both the collected material and the material after TFF1.

[0138] A 10 U / mL hyaluronidase concentration was tested to determine its effect on viscosity and EV count. The following experiments were performed for this purpose:

[0139] ● Prepare a stock solution of 1000 U / mL hyaluronidase in WFI.

[0140] ● Add it to the above 10 mL sample and incubate at 37°C for 30 minutes.

[0141] ● Measuring particle count and viscosity

[0142] ● For viscosity determination, clamp the henke-ject 1mL Luer interface syringe onto the ring support and add a No. 21 blunt needle.

[0143] ● Add a paraffin film to the bottom of the syringe. Add 1 mL of solution to the syringe. Remove the paraffin film and allow it to flow, stopping the timer when the meniscus reaches the last line on the syringe.

[0144] ● Run WFI as a control only

[0145] ● Hyaluronidase stock solution should be stored at -20°C.

[0146] ● 791U / mg

[0147] ● Therefore, add approximately 2 mg (actual: 3 mg) to the tube.

[0148] ● Dissolve in WFI – Add 2.373 mL to dissolve at 1000 U / mL

[0149] ● Add 100uL to 10mL for 10U / mL treatment (untreated: add 100uL WFI)

[0150] ● Mix 15-20 tubes back and forth (inverted tubes)

[0151] ● Add to a 37°C incubator and incubate for 30 minutes.

[0152] ● Take out and measure the viscosity

[0153] The settings can be seen in Table 2.

[0154] Table 2: Experimental setup for collecting / post-treatment of TFF with hyaluronidase

[0155]

[0156] The results showed that the viscosity of samples treated with hyaluronidase was significantly reduced. These results can be applied to... Figure 8 The results showed that EV / mL did not change significantly during treatment. This is important because it demonstrates that compositions containing hyaluronidase can reduce the viscosity of conditioned media and allow for improved downstream processing. This enables chromatography with higher concentrations, fewer filters, and improved upstream processing (improved suspension, nutrient transfer, and recovery).

[0157] Example 2: Hyaluronidase

[0158] Table 3: Representative Hyaluronidases

[0159]

[0160] Hyaluronidases exhibit activity in cleaving the (1→4) bond between N-acetylglucosamine and glucuronide (EC 3.2.1.35), or contain hyaluronidase with (1→3) bond cleaving activity (EC 3.2.1.36) or bacterial hyaluronidase (EC 4.2.2.1). Exemplary hyaluronidase activities are described in this article using the amino acid and nucleic acid sequences encoded by Homo sapiens hyaluronidase-1 isotype 1 precursor (NP_149349.2), bovine hyaluronidase-1 precursor (NP_001017941.1), and Homo sapiens hyaluronidase PH-20 isotype 2 (NP_001167515.1).

[0161] Example 3: Hyaluronidase for downstream processing of extracellular vesicles

[0162] In the standard process, prior to TFF concentration, the conditioned medium from the bioreactor or culture vessel is first clarified by filtration to remove cell debris. The concentrated material is then purified by chromatography, further concentrated by TFF, percolated into a final buffer, aseptically filtered through a 0.2-micron filter, and bottled. However, a significant amount of EV is lost during this process.

[0163] HAase is a novel reagent that can streamline the downstream processing of EVs.

[0164] Hyaluronidase treatment of conditioned medium (CM) from flask cultures or 3D bioreactor cultures significantly reduced pressure buildup / filter contamination during the clarification step, allowing the increased CM volume to be filtered before filter clogging (reaching pressure limits). As CM more easily passes through the filter, EVs do not get trapped, which improves the overall EV yield. Figure 21 This can be done. Figures 20A-B I saw it in the middle. Figure 22 The use of hyaluronidase allows for the use of smaller filter cutoff sizes (e.g., 0.45 µm), which improves purification efficiency. It is noteworthy that filter sizes ranging from 5 µm to 0.2 µm can be used. During bioprocessing prior to TFF and chromatography, a filter size of 0.45 µm is typically used to streamline these unit operations, as well as prior to 0.2 µm (sterile) filtration. This is necessary for optimal performance of downstream processing steps (TFF, chromatography, formulation / filling). It should be noted that small amounts of hyaluronidase added during bioreactor culture at levels that do not affect cell adhesion and growth (approximately 1 mU / mL culture volume) are insufficient to promote downstream purification, and a concentration approximately 100 times higher (100 mU / mL) is optimal for downstream purification. Hyaluronidase can be added to the bioreactor immediately before CM collection or before clarification occurs.

[0165] Figure 23 The study demonstrated that conditioned medium treated with hyaluronidase maintained EV quality properties. For example, expression of four-transmembrane protein markers was maintained. Furthermore, the data showed that hyaluronidase treatment did not cause cell separation from the microcarriers. Figures 24, 25, and 26 show the results at different dosages, incubation times, and temperatures. It was found that 0.1 U / mL balanced the low-pressure buildup in the equilibration filter with high EV yields (equivalent to 5–10 times that of hyaluronidase). Given the high cost of GMP-grade hyaluronidase, it is important to use the minimum amount to achieve good processing and yield.

[0166] In summary, adding hyaluronidase to the filtration process during downstream processing (DSP) of EVs offers the following advantages:

[0167] ● Enhance EV yield through filtration

[0168] ● Improve process efficiency (recycle more EVs)

[0169] ● Enables the use of smaller cutoff filters (tested as low as 0.45µm)

[0170] ● Provides the ability to clarify impurities > 0.45µm (a requirement of DSP) without significant EV loss. The ability to use a smaller cutoff filter enables more efficient TFF because less debris clogs the TFF filter.

[0171] ● This enables the achievement of larger filter loads. Larger filter loads mean that larger volumes of CMs can pass through filters of a given size. Filter costs increase with surface area, therefore smaller filters are more cost-effective.

[0172] These advantages ultimately resulted in a cumulative increase in particle yield of approximately 10-fold (as seen in Figure 18A after the complete purification process), while significantly reducing the cost of GMP preparation operations. The higher yield and lower cost translate into significant cost-effectiveness in therapeutic base development.

[0173] For pressure and load in 3D, the untreated sample is at approximately 32 L / m³. 2When the maximum pressure is reached, the filter needs to be replaced (if the filter is completely contaminated or clogged). If the filter becomes contaminated during processing and the maximum pressure is reached, a second filter needs to be attached, doubling the one-time cost of this unit operation. In this embodiment, only about 15 mL of sample was filtered during a process time of approximately 10 minutes. The pressure versus load curve shows that treatment has a significant impact on improving filtration, where more CM is processed without filter contamination. Furthermore, treatment with any tested reagent V (hyaluronidase) concentration increased the yield by approximately 2.8 times. This effect saturates at 0.1 U / mL. Figure 30 and 31 These results can be seen in the text.

[0174] Example 4: Expansion of human skin fibroblasts

[0175] hMSCs clumpe and increase culture medium viscosity during long-term 3D culture (aimed at maximizing EV collection), leading to culture failure (microcarrier aggregates falling from the solution). The increase in viscosity and aggregation can be mitigated by adding hyaluronidase (Agent V) during culture. In this example, this viscosity accumulation has been demonstrated not only as a challenge for MSCs but also for other primary cell lines such as fibroblasts. Viscosity accumulation has been identified in 3D bioreactor cultures of human skin fibroblasts (HDF), and hyaluronidase has been shown to reduce viscosity and significant microcarrier clumping.

[0176] Figure 31 The experimental setup is shown. HDF was amplified and seeded into rotary flasks containing Corning Synthemax II microcarriers (23K cells / mL, 42 mg microcarriers / mL, total volume 100 mL) in RoosterNourish. On day 5 of EV production (5 days), the medium was replaced with EV-Pro for collection. Cell counts, medium viscosity, and EV counts were monitored over time. Figure 32 The study showed good fibroblast expansion and relatively large microcarrier aggregates, larger than those of MSCs. The culture medium viscosity increased significantly, but the cells remained viable and secreted EVs. Figure 33 illustrates the high cell numbers and high viscosity produced by EVs. The viscosity is higher than that seen in MSC cell cultures.

[0177] On day 10, the viscous medium was treated with reagent V (hyaluronidase) at various concentrations, with TrypLE used as a control. Medium viscosity and EV counts were measured. Reagent V (hyaluronidase) was used at a stock concentration of 100 U / mL. 10% volume of TrypLE and 10x TrypLE were added as test and control, respectively. Similar to MSC medium, HAase reduced the viscosity of the high-viscosity medium in a dose-dependent manner, and the protease TrypLE did not affect viscosity. EV particle counts and particle size profiles were not affected by reagent V. Figure 34 (and 35). This data supports the view that the increased viscosity of the culture medium is not only a phenomenon observed in MSCs, and that agent V can reduce the viscosity of CMs, which is beneficial for long-term microcarrier culture of various cell types. Furthermore, post-collection processing with agent V can improve the yield of filtered EVs.

[0178] In summary, HDF proliferated well in Rooster-Nourish growth medium for 3D microcarrier spinneret cultures, forming large microcarrier aggregates larger than those of MSC 3D cultures. The viscosity of the medium produced by HDF during the EV harvest period indicates that this phenomenon is not specific to MSCs. Hyaluronidase reduced viscosity in a dose-dependent manner, while protease 1xTrypLE did not. These data support that the increased medium viscosity is not limited to MSCs and that Agent V can support long-term microcarrier cultures of multiple cell types.

[0179] Example 5: EV yield in conditioned medium collected from HEK293 cells

[0180] hMSC conditioned medium (CM) contains hyaluronic acid (EV), and significant EV loss occurs when it is filtered for clarification or sterilization. The mechanism appears to be that soluble hyaluronic acid cannot pass through the filter even when viscosity is not an issue, thus trapping EV while contaminating the filter. Treating the medium with HAase breaks down the hyaluronic acid into smaller fragments, allowing them, along with more EV, to pass through the filter.

[0181] This embodiment demonstrates that HAase treatment of the culture medium from ExpiHEK293F cells obtained through a 0.45-micron filter yields a high EV yield. Expi293F cells are a high-protein-expressing HEK 293 cell line sold by Gibco. Culture medium was collected from the cultured Expi293 cells. This experiment determined the effectiveness of Agent V in reducing viscosity and / or increasing EV yield after filtration.

[0182] Figure 36The experimental design is shown. Figure 37 shows the results. Interestingly, HEK medium was found to be non-viscous, but EV recovery increased from 73% to 100% compared to the control.

[0183] Other references

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Claims

1. A method for improving the properties of a liquid cell culture medium, the method comprising introducing hyaluronidase (HAase) into the culture medium.

2. The method of claim 1, wherein the HAase is introduced into the liquid culture medium before the cells are introduced into the liquid culture medium.

3. The method of claim 1, wherein the HAase is introduced into the liquid culture medium after the cells are introduced into the liquid culture medium.

4. The method of claim 1, wherein the HAase is introduced into the liquid culture medium simultaneously with the introduction of cells.

5. The method according to any one of claims 1 to 4, wherein the viscosity of the liquid culture medium decreases by 10% or more when HAase is added.

6. The method according to any one of claims 1 to 5, wherein HAase improves the yield, quality, or processing efficiency of one or more products secreted by said cells in a cell culture.

7. The method according to any one of claims 1 to 6, wherein the cell culture comprises a suspension culture or an adherent culture.

8. The method of claim 7, wherein in the adherent culture, the cells are capable of adhering to plastic, glass, chemically derived surfaces, polypeptides, or polysaccharides.

9. The method according to any one of claims 1 to 8, wherein the liquid culture medium for cell culture is applied to a culture using a microcarrier, a 2D flask container, a bioreactor, a roller flask, a hollow fiber reactor, a stirred tank bioreactor, a vertical wheel bioreactor, or a perfusion bioreactor.

10. The method according to any one of claims 1 to 9, wherein the cell is a human or animal-derived primary cell or stem cell, including embryonic stem cells (ES), adult stem cells, induced pluripotent stem cells (iPSCs), cancer stem cells, mesenchymal stem cells (MSCs), hematopoietic stem cells, hepatocytes, fibroblasts, endothelial cells, blood cells, cancer cells, immune cells, or pancreatic B cells, or derivatives or sublineages of any of these cells.

11. The method according to any one of claims 1 to 10, wherein the cells are Chinese hamster ovary (CHO) cells or CHO-K1 cells or derivatives or sublineages thereof.

12. The method according to any one of claims 1 to 10, wherein the cell is HEK-293 cell or HEK-293T cell or its derivatives or sublineages.

13. The method according to any one of claims 1 to 10, wherein the cell is a mesenchymal stem cell, a pluripotent stromal cell, a mesenchymal stromal cell or a mesenchymal progenitor cell or a derivative or sublineage thereof.

14. The method according to any one of claims 10 to 13, wherein the cells are isolated from: umbilical cord, bone marrow, adipose tissue, adipose stem cells (ASC), adipose tissue, vascular portion of adipose matrix, soft tissue organ, cartilage, perivascular tissue, blood vessel, heart, placenta, ectopic pregnancy tissue, peripheral blood, umbilical cord blood, teeth, or menstrual blood.

15. The method according to any one of claims 1 to 14, wherein the hyaluronidase is used at a concentration of 0.001 U / ml to 500 U / ml.

16. The method of claim 15, wherein the hyaluronidase is used at a concentration of 0.001 U / ml to 0.1 U / ml.

17. The method according to any one of claims 1 to 16, wherein HAase is introduced into the liquid culture medium at a temperature of about 37°C or 25°C (room temperature) and / or in the range of 1°C to 50°C.

18. The method according to any one of claims 1 to 17, wherein the application of HAase to the liquid cell culture medium to improve co-optimized conditions includes changing the stirring rate and the composition of the culture medium, wherein the addition is capable of reducing the viscosity and / or contamination of the cell culture medium and the loss of cell suspension.

19. The method according to any one of claims 1 to 18, wherein the cells in the cell culture are used for the bioproduction of molecules or particles for therapeutic, veterinary, diagnostic, human health, cosmetic and / or therapeutic diagnostic purposes.

20. The method according to any one of claims 1 to 19, wherein the HAase is derived from cells or synthesized synthetically.

21. The method of claim 20, wherein the hyaluronidase is naturally produced by the cells, or wherein the cells are engineered to produce hyaluronidase.

22. The method of claim 21, wherein the cell that produces HAase is one type of cell cultured in the culture medium.

23. The method of claim 21, wherein the cell is a mammalian cell.

24. The method of claim 23, wherein the mammalian cells are derived from mammalian tissues or mammalian organisms.

25. The method according to claim 23 or 24, wherein the mammal is Homo sapiens or Bos taurus.

26. The method according to any one of claims 1 to 25, wherein the HAase activity is derived from a full-length protein or a truncated soluble protein fragment having HAase activity, or wherein the HAase contains one or more mutations compared to a natural HAase molecule.

27. The method of claim 26, wherein the natural HAase contains at least 60% identity with any one of SEQ ID NO: 1-3.

28. The method of claim 26, wherein the HAase is identical to the amino acid sequence associated with any of SEQ ID NO: 1-3.

29. The method according to any one of claims 21 to 28, wherein the engineered cell comprises an expression system synthesized from and / or containing said recombinant nucleic acid.

30. The method of claim 29, wherein the HAase is derived from a coding DNA sequence (cds) that encodes a wild-type, point mutation, deletion mutation, insertion mutation, or codon-optimized expression system to produce the HAase.

31. A method for improving the properties of a liquid cell culture medium, the method comprising culturing cells in the liquid culture medium, removing the cells, and then introducing HAase into the liquid culture medium after cell removal.

32. The method of claim 31, wherein HAase reduces the viscosity of the liquid culture medium by 10% or more.

33. The method of claim 32, wherein HAase improves the yield, quality, or processing efficiency of one or more products secreted by the cells, said one or more products including extracellular vesicles (EVs), exosomes, microvesicles, cellular ghosts, secretory microparticles, or viral particles.

34. The method of claim 33, wherein the product secreted by the cells is a molecule or particle for therapeutic, veterinary, diagnostic, or therapeutic diagnostic purposes.

35. The method according to any one of claims 31 to 34, wherein the liquid culture medium is removed from the cell culture prior to the addition of HAase.

36. The method of claim 35, wherein HAase is added to the liquid culture medium, and then the liquid culture medium is clarified and concentrated.

37. The method of claim 36, wherein the liquid culture medium is clarified by filtration.

38. The method of claim 37, wherein the liquid culture medium is concentrated 2-100 times by percolation, centrifugation or ultracentrifugation.

39. The method according to any one of claims 31 to 38, wherein the HAase is derived from cells or synthesized synthetically.

40. The method of claim 39, wherein the hyaluronidase is naturally produced by the cells, or wherein the cells are engineered to produce hyaluronidase.

41. The method of claim 40, wherein the cell that produces HAase is one type of cell cultured in the culture medium.

42. The method of claim 40, wherein the cell is a mammalian cell.

43. The method of claim 42, wherein the mammalian cells are derived from mammalian tissues or mammalian organisms.

44. The method according to claim 42 or 43, wherein the mammal is Homo sapiens or domestic cattle.

45. The method according to any one of claims 31 to 44, wherein the HAase activity is derived from a full-length protein or a truncated soluble protein fragment having HAase activity, or wherein the HAase contains one or more mutations compared to a natural HAase molecule.

46. ​​The method of claim 45, wherein the HAase is 60% or more identical to the amino acid sequence of any of SEQ ID NO: 1-3.

47. The method of any one of claims 40, wherein the engineered cells comprise an expression system synthesized from and / or containing said recombinant nucleic acid.

48. The method of claim 39, wherein the HAase is derived from a coding DNA sequence (cds) that encodes a wild-type, point mutation, deletion mutation, insertion mutation, or codon-optimized expression system to produce the HAase.

49. The method according to any one of claims 31 to 48, wherein the HAase is provided in an amount between 0.0001 U / ml and 10 U / ml.

50. The method according to any one of claims 31 to 49, wherein the HAase is added directly to the culture medium within the bioreactor.

51. The method according to any one of claims 31 to 50, wherein the HAase is added to the solution containing EV.

52. The method of claim 51, wherein the EV-containing solution is a conditioned medium.

53. The method according to any one of claims 31 to 52, wherein when the HAase is added, the conditioned medium is at a temperature of about 1°C to 50°C.

54. The method according to any one of claims 31 to 53, wherein HAase is added prior to clarification.

55. The method according to any one of claims 51 to 54, wherein the conditioned medium is passed through a filter after exposure to HAase.

56. The method of claim 55, wherein the filter having a pore size between 0.45 µm and 100 µm is used for downstream processing after cell culture.

57. A composition comprising a liquid cell culture medium and an exogenously added hyaluronidase.

58. The composition of claim 57, wherein the liquid cell culture medium comprises cells.

59. The composition according to claim 58, wherein the cell is a mesenchymal stem cell (MSC).

60. The composition according to any one of claims 57 to 59, wherein the cells are attached to a microcarrier, wherein the microcarrier is located in the liquid cell culture medium.

61. The composition according to any one of claims 57 to 60, wherein the cells are attached to microcarriers in a bioreactor, the cells are at a density of 50,000 cells / mL to 5,000,000 cells / mL, wherein the EVs are at a concentration of 1E8 particles / mL to 1E11 particles / mL, and the exogenous hyaluronidase is present at a concentration between 0.0001 U / mL and 100 U / mL.

62. The composition according to claim 60 or 61, wherein the cells have been removed from the liquid cell culture medium.

63. The composition of claim 62, wherein the liquid cell culture medium comprises one or more products secreted by the cells, the one or more products including extracellular vesicles (EVs), exosomes, microvesicles, ghost cells, secretory microparticles, or viral particles.

64. A kit comprising hyaluronidase and liquid cell culture medium.

65. The kit according to claim 64, wherein the HAase is located within the liquid cell culture medium.

66. The kit according to claim 64 or 65, wherein the liquid cell culture medium is capable of being used for cell expansion or collection of conditioned medium.

67. The kit according to any one of claims 64 to 66, wherein the kit further comprises additional components for cell growth, culture, harvesting, or purification of cells or cell products.

68. A composition comprising a conditioned medium derived from MSC cell cultures, wherein the conditioned medium contains EVs, wherein the presence of EVs is between 1E8 and 1E11, and further wherein the conditioned medium contains exogenously added HAase at a concentration of about 0.01 U / mL to 100 U / mL, wherein the conditioned medium has not undergone clarification or filtration.

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