Methods and compositions related to reducing viscosity in cell culture

Abstract

Hyaluronidase can be added to cell culture to reduce viscosity. Contemplated in this invention are methods, compositions, and kits whereby hyaluronidase is added to said cell cultures.

Description

[0001] METHODS AND COMPOSITIONS RELATED TO REDUCING VISCOSITY IN CELL CULTURE CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of U.S. Provisional Application No. 63 / 583,942, filed September 20, 2023, incorporated herein by reference in its entirety. BACKGROUND Basic environmental requirements for cells to grow optimally in culture are: controlled temperature, a substrate for cell attachment, and appropriate growth medium and incubator that maintains correct pH and osmolality. The most important and crucial step in cell culture is selecting appropriate growth medium for in vitro cultivation (Arora, M. Cell Culture Media: A Review. Material Methods 2013;3:175). Mesenchymal stromal cells (MSCs) can be isolated from various tissues such as bone marrow aspirates, fat or umbilical cord blood. These cells have the ability to proliferate in vitro and differentiate into a series of mesoderm-type lineages, including osteoblasts, chondrocytes, adipocytes, myocytes and vascular cells. Due to this ability, MSCs provide an appealing source of progenitor cells which may be used in the field of tissue regeneration for both research and clinical purposes. The key factors for successful MSC proliferation and differentiation in vitro are the culture conditions (Vater, C. et al. Acta Biomaterialia, 2011; Vol. 7, Issue 2, pages 463- 477). The evolution of MSCs culture from small-scale laboratory setups to large-scale bioreactors represents a significant milestone in the biomanufacturing industry (Rowley, J.A., 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 were propagated in multi-well plates and other similar platforms which, while effective for research and small-scale applications, do not meet the demands of commercial scale production. The transition to bioreactor systems has enabled not only the scalable growth of these cells under controlled, repeatable conditions but also adheres to current good manufacturing practices (cGMP), essential for therapeutic applications. This shift was influenced by the need for higher cell yields and more efficient production processes to meet the clinical and commercial demands of cell-based therapies. Bioreactors provide a controlled environment for precise manipulation of culture conditions like oxygen, pH, and nutrient supply, which are critical for maintaining the quality and functionality of MSCs. Furthermore, the use of bioreactors facilitates the implementation of fed-batch and perfusion cultures, enhancing cell expansion capabilities and product consistency. These advancements 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, carrying similar regenerative and immunomodulatory capabilities as their parent cells but with reduced risks and better handling properties. The bioreactor systems initially developed for MSCs are now being adapted to support the production of MSC-EVs, enabling the generation of these potent therapeutic agents in sufficient quantities for clinical research and potential therapeutic use. However, scaling up the production of MSC EVs to meet clinical demands poses significant challenges. As MSCs begin to be used as a producer cell line for EVs, MSC cultures are required to extend in time and scale to maximize productivity (Adlerz, K., Patel, D., Rowley, J.A., Ng. K., Ahsan, T. (2020) Strategies for Scalable Manufacturing and Translation of MSC- derived Extracellular Vesicles, Stem Cell Res, 48, 101978). One specific challenge is that MSC microcarrier suspension cultures fail after extended time during EV production, and this can be exacerbated when cultured in serum free medium. The culture medium experiences an increase in viscosity to the point that the microcarriers coalesce and fall out of solution to the bottom of the bioreactor – essentially ending the culture. Addressing the phenomenon and minimizing the viscosity increase can lead to longer culture times, and enabling longer EV collection for industrialized production processes. The processing and purification of exosomes from MSC conditioned medium collected from flasks or bioreactor can be achieved through several methods, each varying in scalability and efficiency, critical for transitioning from laboratory research to clinical and commercial production. This post-culture processing is commonly called “downstream processing” within the pharmaceutical industry. In small-scale settings, differential centrifugation is a commonly used method to isolate exosomes. This process involves sequential centrifugation steps at increasing speeds to remove cells and debris, followed by ultracentrifugation at higher speeds (e.g., 100,000 g) to pellet the exosomes. Although effective for small volumes, this method is time-consuming and may not be ideal for preserving the functional integrity of exosomes due to the high shear forces involved. Additionally, differential ultracentrifugation typically results in low yields and may not be practical for scaling up to meet clinical demands. For larger scale and more efficient purification, scalable biopharmaceutical production techniques such as Tangential Flow Filtration (TFF) and chromatography are preferred. Prior to the TFF stage the culture medium must be clarified via filtration, often multi-step filtration, to remove cellular debris and contaminating particles that can foul the TFF filter, reducing clearance and yields. This clarification step can begin with a large cutoff filter of several microns, and followed by smaller filters such as 0.8 micron and then 0.45 micron. This clarified conditioned medium is then ready for efficient purification downstream. TFF utilizes a cross-flow mechanism that allows continuous processing of large volumes, effectively concentrating and diafiltrating the exosomes and other soluble proteins from process impurities. This method is advantageous for its scalability, speed, and ability to maintain the biological activity of exosomes due to gentler handling compared to ultracentrifugation. Chromatography, be it size-exclusion or ion exchange chromatography, offers another scalable solution, enabling the separation of exosomes based on size and / or charge. Often after chromatography a second TFF is performed for a final diafiltrations of the product stream into a buffer and to concentrate the solution. There is then a final sterile filtration of 0.2 micron prior to final vialing of the product. These methods are scalable and compatible with GMP manufacturing. One challenge of MSC cell culture medium during concentration via TFF is that the medium can greatly increase viscosity, making it difficult to process, reducing EV yields, and leading to process failures due to high pressures or filter clogging or fouling. Highly viscous solutions also cause challenges in chromatography purification, leading to challenges in this new field of exosome therapeutics. Hyaluronidase is an enzyme that plays a crucial role in the mammalian body by breaking down hyaluronic acid, a major component of the extracellular matrix, thereby facilitating tissue permeability and cellular movement. This enzyme is involved in numerous physiological processes, including wound healing, tissue remodeling, and sperm penetration during fertilization. By degrading hyaluronic acid, hyaluronidase reduces the compactness of the extracellular matrix, enabling the spread of fluids and physical migration of cells through tissues. Hyaluronidase is also 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 primary uses include facilitating subcutaneous fluid infusion (hypodermoclysis), enhancing the absorption and dispersion of drugs in subcutaneous tissues, and promoting the absorption of contrast media in urinary tract angiography (subcutaneous urography). In cosmetic procedures, hyaluronidase is essential 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 underscore hyaluronidase's critical role in improving drug efficacy and addressing complications in various medical and aesthetic procedures. (Jung et al. Arch Plast Surg 2020;47:297-300) Hyaluronidase is widely used in cell isolation from various tissues since hyaluronic acid is a key structural component within the extracellular matrix that gives physical structure to tissues. By adding HAase, with or without other ECM-degrading enzymes such as collagenases or trypsins, researchers have been able to isolate healthy cells from tissues with optimized parameters. Human kidney cells can be obtained from kidney tissue using HAase alongside other enzymes like 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 aids in isolating hepatocytes when combined with collagenase in a perfusion media Drochmans et al. J. Cell Biol. 1975, Vol. 66, No. 1), while in Wharton's jelly from umbilical cords, it is part of a mixed enzymatic-explant method for extracting mesenchymal stem cells (MSCs) (Azandeh et al Journal of Biomedical Science and Engineering Vol.5 No.10, October 2012). Testis cells, particularly gonocytes, are isolated from testis tissue with hyaluronidase used alongside other enzymes (Yang et al. Reproduction, Fertility and Development 22(7) 1057-1065, 2010) and functional mast cells are isolated from bovine lung tissue using hyaluronidase in conjunction with other digestion enzymes (Stahl et al. Journal of Veterinary Medicine, Series B, Vol.43. 1996). HAase has also been used in other areas where hyaluronic acid is used in applications such as in dermal fillers and tissue engineering. Injectable hyaluronic acid (HA) hydrogels have been popularized in facial aesthetics as they provide a long-lasting effect, and low risk of complications. These fillers can be easily removed by the action HAase (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(Pt 2)). HA can be crosslinked to make tissue engineering scaffolds (Sekar MP et al. ACS Biomater Sci Eng. 2023 Jun 12;9(6):3134-3159), and hyaluronidase is often used to degrade these scaffolds to study mechanical properties, or for removing cells for evaluation (Wang et al. PLoS One. 2013 Jun 10;8(6):e56330). Muller et al. (J Immunol Methods. 2014 Sep;411:55-65) tested HAase, along with DNAase and RNAase, for exosome recovery from human plasma, but it was concluded that “attempts at improving exosome recovery from frozen / thawed plasma specimens by enzymatic treatments were neither productive nor cost effective and are not recommended,” thereby teaching away from the present invention. Previous use of HAase in cell culture revolved around breaking down tissue ECM to isolate cells, or to break down crosslinked HA to study the mechanical properties of the underlying material. 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. It discusses how HA, a polysaccharide released by various vertebrate cells, can contaminate EV samples, particularly when using isolation methods like size-exclusion chromatography and tangential flow filtration. The study shows that low and medium molecular weight HA polymers are unexpectedly retained in EV fractions using these filtration-based isolation methods, contributing to the presence of HA contaminants in samples. The research demonstrates that while these methods are effective in removing non-EV-associated proteins, they fail to eliminate HA completely. For instance, medium molecular weight HA (289 kDa) is only partially removed, with significant amounts retained in the final EV fractions. This retention varies depending on the molecular weight of HA and the isolation technique used, suggesting that HA contaminants can inadvertently co-isolate with EVs, affecting the purity and potentially the perceived biological activity of the EV samples. While this manuscript describes HA as a contaminant of MSC-EV preparations, it is not discussed that this can impact cull culture processes or purification yields. In the PCT application "WO2018208670A1," it describes that EVs, such as those derived from umbilical cord, placental tissue, or mesenchymal stem cells (MSC), are covered with complexes of hyaluronic acid and proteoglycans – and the EVs can stick together. Hyaluronidase is used to treat the EVs during processing (at concentrations of 1500 U / mL) to create single EV preparations, to remove the hyaluronic acid and proteoglycan complexes from the surface to expose tethered TGF-β for enhanced therapeutic use. The also believe HAase treatment enhances the isolation and purification of extracellular vesicles (EVs) that have transforming growth factor-beta (TGF-β) tethered to their membrane surfaces by removing hyaluronic acid and proteoglycan complexes from the surfaces of EVs during the centrifugation isolation process. WO2018208670A1 teaches only of hyaluronic acid on the EV surface and does not mention increasing yields during filtration or other pharmaceutical processing steps outside of centrifugation. The PCT application “WO 2017 / 151862 A1” discloses matrix bound nanovesicles and their use. This PCT shows that EVs can be isolated from extracellular matrix (ECM) materials that are derived from various tissues, such as the urinary bladder matrix (UBM), small intestine submucosa (SIS), or other suitable tissues. It uses various enzyme to process the ECM to isolate EVs from the ECM, but not in cell culture or purification applications. The reference "Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles" by Boerre et al (J. Extracellular Ves, 2016) investigates the optimization of extracellular vesicle (EV) isolation from synovial fluid (SF) using hyaluronidase (HYase, also referred to herein as HAase) pretreatment. This reference focuses on the use of hyaluronidase to facilitate the isolation of extracellular vesicles (EVs) from synovial fluid. The prior art addresses the challenge of high viscosity in synovial fluid, caused by the presence of hyaluronic acid (HA), which hampers EV isolation during ultracentrifugation. Hyaluronidase is employed in this context to degrade HA and improve the recovery of EVs, specifically CD44+ vesicles, from this naturally occurring biological fluid. The methods outlined in the prior art do not address the specific challenges or requirements associated with cell cultures, bioreactor environments, or the industrial production of EVs. For the industrialized production of EVs, there is a need for production using scalable production technologies such as bioreactors, and scalable purification systems like filtration, TFF and chromatography. The existing art lacks methods for extending culture time in bioreactors for EV producing cells, such as by minimize viscosity build up over time. There is also the need to purify these scalably produced EVs using traditional biopharmaceutical technologies. By enabling the scalable production and purification of EVs, more product can be produced, more product can be recovered during purification, fewer process failures will occur, and overall products can have much higher manufacturability leading to lower cost of goods per unit volume. SUMMARY Disclosed herein is a method of improving properties of a liquid cell culture medium, the method comprising introducing hyaluronidase (HAase) into the medium. Also disclosed herein is a method of improving properties of liquid cell culture media, the method comprising culturing cells in the liquid medium, removing said cells, and then introducing HAase to the liquid medium after cells are removed. Further disclosed herein is a composition which comprises liquid cell culture media and hyaluronidase. Also disclosed herein is a kit comprising liquid cell culture media and hyaluronidase. Further disclosed herein is a composition comprising conditioned medium derived from an MSC cell culture, wherein the conditioned media comprises EVs, wherein between 1e8 and 1e11 EVs are present, and further wherein the conditioned medium comprises exogenously added HAase at a concentration of about 0.01 to 100 U / mL, wherein the conditioned medium has not undergone clarification or filtration. DESCRIPTION OF DRAWINGS Figure 1A-B exemplifies a typical EV production test run in which microcarrier aggregation and MSC culture fouling may be disrupted under selective media and bioreactor conditions, i.e., EV Collection Medium B. Figure 1A exhibits simplified upstream bioprocess schemes where a single vial of MSCs first expands on a two-dimensional T-225 CellBIND flask, which then provides seeding material for culture on microcarriers in Ambr250® culture vessels on Day 0 of EV preparation. After sufficient expansion, RoosterNourish medium is exchanged on Day 5 for media (A or B) that is specifically formulated for an EV collection phase. Figure 1B shows photographic images of a time course of representative Ambr250® culture bioreactors containing live hMSCs. Day 5 images demonstrate effect of media switching from RoosterNourish expansion medium to EV collection media (A, B, and B + dextran sulfate). The microcarriers aggregate and foul the MSC culture with Collection Medium B or even B + dextran sulfate, which fails to reduce clumping or offset loss of the EV-conditioned medium as intended. Cells maintained in Medium A do not show substantial microcarrier aggregation, whereas microcarriers with cells in Medium B continue aggregate and precipitate to the bottom of the flask, fouling the culture through Day 10. Figure 2 shows that the viscosity index dramatically increased by Day 10 of cells maintained in EV Collection Medium B. High media viscosity presents unique challenges to microcarrier cell culture and to collection of secreted cell culture products. The phenomenon is concomitant with culture fouling of cells grown on microcarriers. Cell culture viscosity was measured and recorded as the viscosity index, or [(viscosity timesample) - (viscosity timewatercontrol)] / (viscosity timewater control)]. Viscosity time is the total migration time for 1 mL of liquid solution to proceed through a standard 21 -gauge blunt needle from a loaded syringe, attached to a ring stand. Viscosity Index is controlled to account for varying relative humidity and atmospheric conditions.

[0002] Figure 3A-C shows the effect of hyaluronidase on culture viscosity. Figure 3 A shows that post-harvest media viscosity can be selectively ameliorated by treatment with 500 mU / mL hyaluronidase (HYAL)[ bovine HYAL (NP_001017941.1; 1.0 mU / mL, Millipore Sigma, Saint Louis, Missouri)], in contrast with Collagenase, Accumax, or TrypLE, which exert no effect on viscosity index. Figure 3B demonstrates dose response of HYAL (0 to 100 U / mL) on viscosity index of MSC-conditioned medium in comparison with TyrpLE and Trypsin. Figure 3C validates that treatment of MSC-conditioned medium across a range of HYAL concentrations or cell detachment formulations (1x TrypLE, lx Trypsin) does not affect particle counts of EVs as measured by NTA (nanoparticle tracking analysis).

[0003] Figure 4 shows dose response on viscosity index across concentrations of recombinant, HIS-tagged human HYAL (NP 001167515.1; 0.1, 1.0, or lOmU / mL, ACRO; rPH20 from ACRO Biosystems, Newark, Delaware) affecting EV-conditioned collection medium after addition on Day 7, compared with bovine HYAL (NP_001017941.1; 1.0 mU / mL, Millipore Sigma, Saint Louis, Missouri) or untreated. Viscosity indices collected via Days 10 and 12 depict comparable per unit activity between preparations of human recombinant and bovine testes isoforms of HYAL.

[0004] Figure 5 shows that hyaluronidase does not seem to have a negative impact on MSC morphology / viability during flask culture, and cells remain adherent to culture surfaces, even up to 500 U / ml.

[0005] Figure 6A-B shows that cells fall off microcarriers when 250 U / ml of hyaluronidase is used. Figure 6A and 6B shows proliferation of cells with and without hyaluronidase treatment.

[0006] Figure 7A-C illustrates that methods involving addition of HYAL may require special optimization between variables of HYAL dose, and culture conditions. Figure 7 A shows images of labeled hMSCs attached to microcarriers (me) grown in miniature bioreactors under varying concentrations of HYAL between days 3 and 7 after seed culture transfer. MSFv15 is a unique cell culture medium formulation. All concentrations of HYAL shown (10 U / mL to 250 U / mL) demonstrate that these concentrations do not support healthy cell cultures, and cells no longer attach to microcarriers and poor cell growth occurs. Figure 7B shows images of healthy cultures at greatly reduced HYAL concentration of 0.001 U / mL ( 1 mU / mL) as can be seen by the labeled hMSCs maintaining attachment to microcarriers (mc) grown in miniature bioreactors under a ”microdose” of 1.0 mU / mL (- / +) HYAL between days 3 and 12 after seed culture transfer. Figure 7C demonstrates activity of HYAL via Viscosity Index in conditioned media collected from cells shown in Figure 7B. The dose of 1.0 mU / mL HYAL is sufficient to both reduce media viscosity as well as allow attachment of hMSCs under these culture conditions. Figure 8 shows that adding hyaluronidase every 3 days still leads to some cells coming off of the carriers. Figure 9 shows that viscosity is significantly reduced for samples treated with hyaluronidase, both harvested cells and TFF concentrated cells. Figure 10A-C demonstrates that treatment of exemplar adherent mammalian cells (hMSCs) in 2D planar culture does not damage the cells across a time course when administered varying doses of hyaluronidase (10-250 U / mL HYAL), including a very high dose (250 U / mL). Figure 10A shows a representative experiment to determine the toxicity of hyaluronidase to MSCs in culture. HYAL was added to cells on Day 3 after seeding, and cell health was monitored (Image at 10X) via morphology to day 7. Spent medium analysis for Viscosity Index was carried out in parallel validated activity of HYAL. Levels tested were 0, 10, 30, 100, 250 U / ml hyaluronidase. It was found that hyaluronidase did not have a negative impact on MSC morphology / viability during flask culture, and the cells remained adherent to culture surface, even up to 250 U / ml. Figure 10B depicts increasing cell density in dose range of HYAL at days 3 and 5, from cells shown in Figure 5A, indicating no effect of HYAL on growth at even 250 U / mL. Figure 10C depicts maintained % viability in dose range of HYAL at days 3 and 5, from cells shown in Figure 11A, indicating no effect of HYAL on viability at even 250 U / mL. Figure 11A-B demonstrates representative experiments where treatment of EV- conditioned media via a broad concentration range (0 to 100 U / mL) of HYAL does not adversely affect their biological activity on cell culture assays of wound healing (Figure 11A) or angiogenesis (Figure 11B). CM samples were clarified, treated with HYAL, and ultracentrifuged. The EV pellet was resuspended in the assay media and dosed at the same number of particles for each assay. More specifically, 14A shows potency of EVs derived from day 10 hMSC microcarrier culture and subsequently treated with a range of HYAL concentrations (0.0 to 100 U / mL) or trypsin (Tryp lx). Enzyme-treated EVs were applied to scratched HUVEC monolayers, and scratch closure was measured. Note effect of trypsin on diminution of wound closure activity, and lack of HYAL-related inhibition of EV bioactivity. Bars, SD. Figure 11B shows representative images and quantified angiogenic potency of EVs derived from day 10 hMSC microcarrier culture in Ambr250 and subsequently treated with a range of HYAL concentrations (O.lmU / mL to 100 U / mL) or trypsin. EVs were applied to HUVECs atop Cultrex gel layer and incubated overnight. Total mesh area was analyzed using Angiogenesis Analyzer in Image!. Note that active HYAL concentrations (0.0001 to 100 U / mL) did not hinder the angiogenic mesh forming capability of HUVECs. Bars, SD.

[0007] Figure 12A-B demonstrates that cells grown in Ambr bioreactors on microcarriers can be prevented from culture fouling by timely addition of hyaluronidase (HYAL) at specific “microdosing” concentrations of as low as 1.0mU U / mL. Figure 12A shows an upstream EV bioprocess flow diagram with MSCs grown in Ambr250® flasks, employed for optimization of HYAL concentrations and bioreactor physical process parameters. Figure 12B shows representative images across a dose and time course of hMSC microcarrier culture in Ambr250(r) flasks, where Day 5 was 5 days post-inoculation of seed culture into flask with microcarriers, when expansion medium was exchanged with 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. Note microcarrier precipitation and culture fouling on Days 7-10 under concentration conditions of < 1.0mU / mL, but no visible microcarrier aggregation and fouling with the higher dose of 1 mU / mL (0.001 U / mL).

[0008] Figure 13A-C depicts how HYAL media supplementation at concentrations between “microdoses” of 0.01 mU / mL and 1.0 mU / mL affects culture viscosity as well as yield. Yield is enhanced when hMSCs are cultured in microcarrier flasks with an active, optimized concentration of HYAL (1.0 mU / mL), and gross particle size and distribution of a secreted cellular EV / exosome product is maintained. Figure 13A demonstrates that HYAL administered at “optimal” concentration (1.0 mU / mL) is sufficient to rapidly reduce EV collection medium viscosity, in concert with prior observations of reversal of microcarrier culture fouling (Figure 8). Bars, SD. Figure 13B demonstrates yield of a secreted cellular product (extracellular vesicles, EVs) into the conditioned culture medium is enhanced approximately 3-fold by treatment with optimal HYAL (1.0 mU / mL) at Day 6. Figure 13C shows that the particle size distribution of EVs collected from this experiment are from conditioned media are mostly alike in median size and distribution via samples analyzed with a NanoSight NTA instrument. Figure 14A-C shows a range of agitation conditions tested in spinner flasks. 14A shows versus the singular condition tested in Ambr250. Figure 14A shows a range of agitation conditions tested in spinner flasks versus the singular condition tested in Ambr250. 14B shows that HYAL treatment is effective across a range of agitation conditions. Excessive agitation (6800 W / m3) affects cell growth and attachment, and thus the medium does not become viscous. Data are acquired on culture Day 10. NM = not measured.14C shows cell attachment under conditions from Fig.14B. Cells remain attached in the range 1.6-36 W / m3independent of HYAL treatment. NM = not measured. Figure 15A-B shows an experiment that identifies an appropriate HYAL concentration, temperature, and incubation time for treatment of conditioned medium produced at a large-scale (15L STR). 15A shows that 200mL samples of harvested conditioned medium viscosity are reduced by treatment with 100 mU / mL HYAL in ~10 minutes at room temperature but incubated up to 37°C. Surprisingly, it appears that HYAL is active not merely in cell culture incubation at 37°C, but also at room temperature. Error bars are SD. Figure 15B shows results from the bulk 15L harvested conditioned medium treated with 100 mU / mL HYAL at room temperature for 10min. Error bars are SD. Figure 16A-D shows an experimental bioprocess of where active HYAL (- / +) can be used to reduce viscosity of an EV Collection Medium, after cell culture and post-harvest, showing that HYAL (100 mU / mL x 30 min. at room temperature) can improve aspects of downstream processing (DSP) of a secreted exosome / EV cellular product. 16A depicts DSP schemes compared to test 1.2L volumes of hMSC conditioned bioreactor medium (CM) treated with HYAL 100 mU / mL (- / +). 16B shows that treatment of CM with HYAL decreases viscosity ~4- fold. 16C and 16D show that the HYAL treatment leaves EV particle concentration (16C) and size distribution (16D) unaffected. Figure 17A-B show filtration in treated vs. untreated samples. Figure 17A shows large- pore size depth filter, compares HYAL (0.1 U / mL) treated and untreated conditioned media subjected to Filtration 1. The untreated sample over-pressurizes the filter, which had to be replaced with a larger unit of additional cost. The HYAL-treated sample did not experience this problem. Figure 17B shows small-pore size depth filter, compares HYAL treated (- / +) conditioned media subjected to Filtration 2 prior to chromatography. The untreated sample over- pressurizes the filter, wherein a new filter of additional cost must be replaced and operated at a lower flowrate, extending the time of processing to >15 minutes. The HYAL-treated sample does not experience this issue, where filtration completes in around 5 minutes. Figure 18A-C shows cumulative particle recovery in HYAL treated samples. Figure 18A demonstrates that HYAL treatment according the DSP scheme dramatically improves % process recovery of EVs on a cumulative basis, with 52% recovered (HYAL treated) vs. 4% recovered (HYAL untreated) by the end of the process. Figure 18B shows in vitro scratch wound closure activity of EVs is maintained post downstream processing conducted with and without HYAL treatment. Experiment is conducted using a monolayer of HUVEC cells. Control samples utilize basal medium (Neg. Ctrl) or complete medium (Pos. Ctrl). Experimental samples were taken after the Filtration 3 (final) step of DSP. Bars represent the mean of N=6 (Neg. Ctrl), N=4 (Untreated), N=7 (Pos. Ctrl, HYAL Treated) replicates conducted per group. Error bars represent SD. *, p < 0.05 via ANOVA with Dunnett’s multiple comparisons test. Figure 18C shows enzymatic activity of CD73 present on EVs is maintained post downstream processing conducted with and without HYAL treatment. (Left) Assay controls include recombinant human CD73 (rhCD73) untreated (Pos. Ctrl) or heat-inactivated (Neg. Ctrl). (Right) Samples tested for Untreated and HYAL treated are post-Filtration 3 step (Fig. 18A). Bars represent the mean of N=2 experiments conducted per group. Figure 19A-C shows maintenance of EV marker expression CD63 (A), CD9 (B), and CD81 (C) before and after treatment with HYAL. Western blot results were obtained using ProteinSimple Jess capillary western blot system. Samples were taken after the Filtration 3 (final) step of DSP. Figure 20A-B shows that treatment with hyaluronidase (also referred to herein as “Agent V”) leads to minimal pressure build-up during filtration. Figure 21 shows that hyaluronidase increases particle yield through clarification. Figure 22 shows that hyaluronidase enables use of smaller filter cutoff (0.45µm), improving purification capability. Figure 23 shows that treatment of conditioned medium with Agent V maintains EV quality attributes. For example, tetraspanin marker expression is maintained. Figure 24 A and B show that pressure improvement increases with Agent V (hyaluronidase) dose, reaching saturation ~0.5U / mL. Maximum yield benefit observed at 1.0 U / mL. (A) shows loading pressure, (B) shows particle yield. Figure 25A-B shows that increasing incubation time to 30 minutes improved the pressure profile. Even immediate addition of Agent V improves filtration. Yield benefit is saturated at this treatment condition. Figure 26A-B shows temperature information for 4°C, room temperature (RT), and 37°C. (A) shows pressure, (B) shows particle yield. Figure 27A-D shows no discernable trends in size distribution depending on Agent V treatment parameters after filtration. (A) process; (B) dose response; (C) incubation time; (D) temperature. Figure 28 shows pressure versus loading results for hyaluronidase (Agent V) treated vs. untreated samples. Figure 29A-B shows particle yield results for treated vs. untreated samples. (A) shows particle concentration; (B) shows particle yield. Figure 30 shows addition of hyaluronidase during downstream processing. Figure 31 shows the experimental design for testing human dermal fibroblasts using hyaluronidase to determine effectiveness of hyaluronidase to reduce viscosity and significant microcarrier clumping. Figure 32 shows HDF microcarrier aggregated substantially, even more so than MSC cultures. Representative images 4x. Figure 33A-D shows high cell counts and high viscosity with EV production in HDFs. The culture is much more viscous than MSC cultures. (A) shows sampled cell density. (B) shows particles / mL. (C) shows viscosity index. (D) shows frequency. Figure 34 shows post-harvest treatment using hyaluronidase titration in HDFs. Figure 35A-B shows HDF post-harvest viscosity treatment. (A) shows viscosity index at various concentrations of hyaluronidase. (B) shows percent recovery of EVs during a filter test. Figure 36 shows the experimental design for determining if exposure of HEK cells to hyaluronidase can affect yield of EVs. Figure 37A-B shows that HEK medium was not viscous, but EV recovery was still significantly increased from about 73% to about 100%. (A) shows viscosity index of control and conditioned medium. (B) shows filter test and percent particle recovery of EVs using different concentrations of hyaluronidase. DETAILED DESCRIPTION General Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. By “about” is meant within 10% of the value, e.g., within 9, 8, 8, 7, 6, 5, 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for 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,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps. As used in the specification and claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof. As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient. The term “extracellular material” (also referred to as “EM”) refers to cell-derived material, including extracellular vesicles (EVs), that are present in eukaryotic fluids, including blood, lymph, urine, saliva and conditioned medium of cell cultures. This extracellular material can be derived from cellular endosomal and plasma membranes, and can comprise secretory components. Examples of these components comprise, but are not limited to, nucleic acids such as DNAs, RNAs (including miRNA), proteins, polypeptides, carbohydrates, lipids, and small molecules. Extracellular material can be cell-derived small vesicles, and can include exosomes, microvesicles, ectosomes, shedding vesicles, membrane vesicles, plasma membrane vesicles or other membrane-bound assemblies, and apoptotic bodies, viruses, and cells. In certain embodiments, the extracellular material can comprise, consist, or consist essentially of extracellular vesicles. The term “cell” as used herein also refers to individual cells, cell lines, primary culture, or cultures derived from such cells unless specifically indicated. A “culture” refers to a composition comprising isolated cells of the same or a different type. Living cells can be found in cell culture. The term "mammalian cell" means any cell from or derived from any mammal (e.g., a human, a hamster, a mouse, a green monkey, a rat, a pig, a cow, or a rabbit. In some embodiments, the mammalian cell can be, e.g., an immortalized cell, a differentiated cell, or an undifferentiated cell. The term “multipotent stem cell” means a cell which is capable of giving rise to multiple different types of cells. The term “mesenchymal stem cell” means a stem cell originally derived from the mesenchyme. The term refers to a cell which is capable of differentiating into at least two or more of an osteoblast, a chondrocyte, an adipocyte, or a myocyte. Mesenchymal stem cells (MSC) are isolated from any type of adult tissue. Typically mesenchymal stem cells are isolated from bone marrow, adipose tissue, umbilical cord, or peripheral blood. In a preferred aspect, MSCs are obtained from bone marrow or lipoaspirates, which are obtained from adipose tissue. The term “multipotent” or “pluripotent” also encompasses 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 certain embodiments, the multipotent or pluripotent stem cells of the disclosure are mesenchymal stem cells. The term “population” with reference to the multipotent and mesenchymal stem cells described herein refers to a plurality of cells which are molecularly identical or highly similar. A population can be isolated from a human tissue or grown and expanded in culture. Any population provided herein can “consist essentially” of the recited cells such as multipotent stem cells or mesenchymal stem cells. As used herein “consist essentially” means that the cell population is substantially pure from a cellular composition perspective. For example, the cells can be greater than about 80%, 90%, 95%, 97%, 98%, or 99% pure. The term “unstimulated” or “naive” refers to a cell population that has been untreated, unpolarized, or uninduced by the methods of this disclosure. Primary isolated mesenchymal stem cells that are fresh or frozen are considered unstimulated or naive. A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative." The term "substantially free" means a composition (e.g., a liquid culture medium) that is at least or about 90% free (e.g., at least or about 95%, 96%, 97%, 98%, or at least or about 99% free, or about 100% free) of a specific substance (e.g., a mammalian cell or microcarriers). The term "culturing" or "cell culturing" means the maintenance or growth of a mammalian cell in a liquid culture medium under a controlled set of physical conditions. The term "liquid culture medium" means a fluid that contains sufficient nutrients to allow a mammalian cell to grow in the medium in vitro. For example, a liquid culture medium can contain one or more of: amino acids (e.g., 20 amino acids), a purine (e.g., hypoxanthine), a pyrimidine (e.g., thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc, selenium, and other necessary trace metals, and sodium bicarbonate. A liquid culture medium may contain serum from a mammal. In some instances, a liquid culture medium does not contain serum or another extract from a mammal (a defined liquid culture medium). A liquid culture medium may contain trace metals, a mammalian growth hormone, and / or a mammalian growth factor. Non-limiting examples of liquid culture medium are described herein and additional examples are known in the art and are commercially available. The term "microcarrier" means a particle (e.g., an organic polymer) that has a size of between 20 µm to about 1000 µm that contains a surface that is permissive or promotes attachment of a mammalian cell (e.g., any of the mammalian cells described herein or known in the art). A microcarrier can contain one or more pores (e.g., pores with an average diameter of about 10 µm to about 100 µm). Examples of microcarriers are known in the art. A microcarrier can contain, e.g., a polymer (e.g., cellulose, polyethylene glycol, or poly-(lactic-co-glycolic acid)), for example. The term "animal-derived component free liquid culture medium" means a liquid culture medium that does not contain any components (e.g., proteins or serum) derived from an animal. The term "serum-free liquid culture medium" means a liquid culture medium that does not contain animal serum. The term "serum-containing liquid culture medium" means a liquid culture medium that contains animal serum. The term "chemically-defined liquid culture medium" means a liquid culture medium in which substantially all of the chemical components are known. For example, a chemically- defined liquid culture medium does not contain fetal bovine serum, purified bovine serum albumin, or purified human serum albumin, as these preparations typically isolated from blood and contain a complex mix of albumins and lipids. Chemically defined medium may contain albumins that are recombinantly produced.. The term "protein-free liquid culture medium" means a liquid culture medium that does not contain any protein (e.g., any detectable protein). “Viscosity” is the magnitude of internal friction within a fluid, i.e. intermolecular friction between neighboring particles that are moving at different velocities. The size, shape and magnitude of intermolecular forces of particles within a fluid determine its viscosity. As cell cultures proliferate, metabolic activity and other processes can be expected to increase the number of solute particles in culture media, thereby increasing its viscosity. Cell culture viscosity was measured and recorded as the viscosity index, or [(viscosity timesample) - (viscosity timewatercontrol)] / (viscosity timewater control)]. Viscosity time is the total migration time for 1 mL of liquid solution to proceed through a standard 21-gauge blunt needle from a loaded syringe, attached to a ring stand. Viscosity Index is controlled to account for varying relative humidity and atmospheric conditions. Compositions, Kits, and Methods of Use Disclosed herein are methods, kits, and compositions for extending the useful life of a cell culture by decreasing viscosity of the cell culture medium, thus making the cell culture more productive. Also disclosed are methods, kits, and compositions for improving the culture medium quality thus enabling easier and high yielding purification of biological products with the culture medium, such as EVs. The culture medium quality can be improved in terms of yield, quality, or processing efficiency. This improvement can be by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or 2, 3, 4, 5, 6, 7, 8, or 10 fold or more compared to a control in which conditions are the same but no HAase is used.

[0009] The culture medium quality can be improved by introducing hyaluronidase into the cell culture medium, either before, during (concurrently with) or after culture. Hyaluronidase can be added to cell culture medium in a variety of culture configurations, or even added after the cells have been removed from the medium for purification purposes. For example, the cells can be grown on a microcarrier in a bioreactor configuration, or in a flask. This invention is also drawn to improving purification of cell culture medium containing EVs, for example, which have been grown in bioreactors or flasks, by reducing pressure build-up during filtration, resulting in greater yields. These uses, and others, are discussed in detail below.

[0010] As used herein, “hyaluronidase” refers to a class of hyaluronan degrading enzymes. The hyaluronidase used with the present invention can be naturally occurring or can be recombinant. It can be synthetically manufactured or can be naturally derived. It is noted that hyaluronidase is alternatively referred to herein as “HAase,” “HYAL,” or “Agent V.” All of these are defined as being the same as “hyaluronidase” which is defined in detail below. One of skill in the art will appreciate how to obtain and use hyaluronidase with the methods and compositions disclosed herein. Examples of hyaluronidase which can be used with the present invention can be found in Table 3 (SEQ ID NOS: 1-3).

[0011] Hyaluronidases include bacterial hyaluronidases (EC 4.2.2.1 or EC 4.2.99.1), hyaluronidases from leeches, other parasites, and crustaceans (EC 3.2.1.36), and mammaliantype hyaluronidases (EC 3.2.1.35). Hyaluronidases include but not limited to, murine, canine, feline, leporine, avian, bovine, ovine, porcine, equine, piscine, ranine, bacterial, and any from leeches, other parasites, and crustaceans. Examples of hyaluronidases can be found, for example, in European Patent EP2833905B1, which is herein included by reference in its entirety for its teaching concerning hyaluronidase. Types and classifications of hyaluronidase can be found in Jung H. Hyaluronidase: An overview of its properties, applications, and side effects. Arch Plast Surg. 2020 Jul;47(4):297-300. doi: 10.5999 / aps.2020.00752, which is also incorporated by reference in its entirety for its teaching concerning hyaluronidase. Specifically, HAase can be derived from a mammalian source, such as Homo sapiens or Bos taurus. The HAase can be naturally occurring or can comprise one or more mutations compared to a naturally occurring, or native, HAase. HAse activity can be derived from a full-length protein, or a truncated soluble protein fragment with HAase activity, or wherein the HAase comprises one or more mutations compared to a native HAase molecule. For example, the HAase found in SEQ ID NOS: 1-3 can comprise 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% identity to a native HAase, or any amount between, below, or above these percentages. These mutations can be in either the amino acid of the HAase or in the nucleic acid encoding the HAase. These mutations can comprise, but are not limited to, coding DNA sequences (cds) encoding wild type, point mutated, deletion mutated, insertionally mutated, or codon-optimized expression systems to produce the HAase. HAase, as used with the methods and compositions disclosed herein, can be naturally produced by the cell, a cell can be engineered to produce hyaluronidase. For example, the HAase can be from cultured cells in the media so that HAase need not be exogenously added. The cell producing HAase can naturally produce HAase, or can be engineered to produce it. When engineered, the cell can comprise an expression system that is synthesized from and / or comprised of recombinant nucleic acids which encode the HAase. Hyaluronidase can be used with various types of cells and various types of cell cultures, including adherent and suspension cultures. In adherent cell culture, cells are grown while attached to a substrate as monolayers. In suspension cell culture, cells are free floating in the culture medium. An example of an adherent cell culture is the use of microcarriers. Microcarriers are well known in the prior art. Microcarriers can be made from a number of different materials. In some embodiments, the microcarriers are made with a non-biodegradable material, such as cellulose, DEAE-dextran, hydroxylated methacrylate, polyacrylamide, polystyrene, plastic, glass, ceramic, and silicone. In some embodiments, the microcarriers are made with a biodegradable materials such as collagen, alginate, dextran, gellan gum and gelatin. These microcarrier materials, along with different surface chemistries, can influence cellular behavior, including morphology and proliferation. Surface chemistries can include extracellular matrix proteins, recombinant proteins, peptides, and positively or negatively charged molecules. For example, the microcarriers can be coated with collagen, fibronectin, pronectin, matrigel, laminin, vitronectin, or e-cadherin. The microcarriers can be positively charged, or surface modified with peptide conjugate such as nanofiber or thermos reversible / responsive surface such as poly N-isopropylacrylamide (poly- NIPAAM). In some embodiments, the microcarriers are magnetic beads. Microcarriers preferably have density that allows them to be maintained in suspension with gentle stirring. For example, the microcarriers can have a specific gravity between 1.015 g / cc and 1.035 g / cc. Microcarrier cell culture is typically carried out in spinner flasks, although other vessels such as rotating wall microgravity bioreactors or fluidized bed bioreactors can also support microcarrier-based cultures. Several types of microcarriers are available commercially including alginate-based (GEM™, Global Cell Solutions), dextran-based (Cytodex™, GE Healthcare), collagen-based (Cultispher™, Percell), and polystyrene-based (SoloHill Engineering) microcarriers. They differ in their porosity, specific gravity, optical properties, presence of animal components, and surface chemistries. In this specific application, cells are cultured in bioreactors on microcarriers specifically for the production of EVs. Cells, such as MSCs, are first expanded on microcarriers during a growth phase, and then the cultures are used to produce EVs or other extracellular materials that are often used in therapeutic development. The medium to collect EVs in is typically, but not always, a different medium than the cells were grown in. The EVs are produced by the cultured cells into the EV collection medium, and the longer the cells can be cultured the more EVs are produced. During extended culture of MSCs, and other cells, in order to extend the EV production phase, the cell culture medium can become viscous, impacting the quality of the culture. It is herein disclosed that adding hyaluronidase to the culture medium allows for a control in medium viscosity, enabling longer lived cultures and more productivity. It is also disclosed that hyaluronidase can be added to the conditioned medium after harvest to aid in purification, thus enhancing yields. Many novel methods of improving cell culture medium for enhanced production, or enhanced purification, of EVs and other cell produced materials, via the addition of hyaluronidase at the optimal time, at the optimal concentration, and in an optimal pattern (single vs repeated dosing) have been discovered and are described within. One of skill in the art will appreciate the types of cells which can be used with the cell culture techniques described above. The cells for use with the compositions disclosed herein described herein can be any cells capable of culture, and can include living, viable cells. The cells can be modified. For example, the cells can be genetically modified to effect a permanent or temporary change in physicochemical features, biodistribution, pharmacokinetics, pharmacodynamics, or biological functions of said extracellular material. In some embodiments, the disclosed composition contains stem cells or progenitor cells. Pluripotent stem cells, adult stem cells, blastocyst-derived stem cells, gonadal ridge-derived stem cells, teratoma-derived stem cells, totipotent stem cells, multipotent stem cells, oncostatin- independent stem cell (OISCs), embryonic stem cells (ES), embryonic germ cells (EG), and embryonic carcinoma cells (EC) are all examples of stem cells. Stem cells can have a variety of different properties and categories of these properties. For example in some forms stem cells are capable of proliferating for at least 10, 15, 20, 30, or more passages in an undifferentiated state. In some forms the stem cells can proliferate for more than a year without differentiating. Stem cells can also maintain a normal karyotype while proliferating and / or differentiating. Stem cells can also be capable of retaining the ability to differentiate into mesoderm, endoderm, and ectoderm tissue, including germ cells, eggs and sperm. Some stem cells can also be cells capable of indefinite proliferation in vitro in an undifferentiated state. Some stem cells can also maintain a normal karyotype through prolonged culture. Some stem cells can maintain the potential to differentiate to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm) even after prolonged culture. Some stem cells can form any cell type in the organism. Some stem cells can form embryoid bodies under certain conditions, such as growth on media which do not maintain undifferentiated growth. Some stem cells can form chimeras through fusion with a blastocyst, for example. Some stem cells can be induced or transformed from non- stem cells by genetic or chemical means. In some embodiments, the disclosed composition comprises a cell other than a stem cell. The adult human body produces many different cell types. These different cell types include, but are not limited to, Keratinizing Epithelial Cells, Wet Stratified Barrier Epithelial Cells, Exocrine Secretory Epithelial Cells, Hormone Secreting Cells, Epithelial Absorptive Cells (Gut, Exocrine Glands and Urogenital Tract), Metabolism and Storage cells, Barrier Function Cells (Lung, Gut, Exocrine Glands and Urogenital Tract), Epithelial Cells Lining Closed Internal Body Cavities, Ciliated Cells with Propulsive Function, Extracellular Matrix Secretion Cells, Contractile Cells, Blood and Immune System Cells, Sensory Transducer Cells, Autonomic Neuron Cells, Sense Organ and Peripheral Neuron Supporting Cells, Central Nervous System Neurons and Glial Cells, Lens Cells, Pigment Cells, Germ Cells, and Nurse Cells. Cells of the human body include Keratinizing Epithelial Cells, Epidermal keratinocyte (differentiating epidermal cell), Epidermal basal cell (stem cell), Keratinocyte of fingernails and toenails, Nail bed basal cell (stem cell), Medullary hair shaft cell, Cortical hair shaft cell, Cuticular hair shaft cell, Cuticular hair root sheath cell, Hair root sheath cell of Huxley's layer, Hair root sheath cell of Henle's layer, External hair root sheath cell, Hair matrix cell (stem cell), Wet Stratified Barrier Epithelial Cells, Surface epithelial cell of stratified squamous epithelium of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cell (stem cell) of epithelia of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, Urinary epithelium cell (lining bladder and urinary ducts), Exocrine Secretory Epithelial Cells, Salivary gland mucous cell (polysaccharide-rich secretion), Salivary gland serous cell (glycoprotein enzyme-rich secretion), Von Ebner's gland cell in tongue (washes taste buds), Mammary gland cell (milk secretion), Lacrimal gland cell (tear secretion), Ceruminous gland cell in ear (wax secretion), Eccrine sweat gland dark cell (glycoprotein secretion), Eccrine sweat gland clear cell (small molecule secretion), Apocrine sweat gland cell (odoriferous secretion, sex-hormone sensitive), Gland of Moll cell in eyelid (specialized sweat gland), Sebaceous gland cell (lipid-rich sebum secretion), Bowman's gland cell in nose (washes olfactory epithelium), Brunner's gland cell in duodenum (enzymes and alkaline mucus), Seminal vesicle cell (secretes seminal fluid components, including fructose for swimming sperm), Prostate gland cell (secretes seminal fluid components), Bulbourethral gland cell (mucus secretion), Bartholin's gland cell (vaginal lubricant secretion), Gland of Littre cell (mucus secretion), Uterus endometrium cell (carbohydrate secretion), Isolated goblet cell of respiratory and digestive tracts (mucus secretion), Stomach lining mucous cell (mucus secretion), Gastric gland zymogenic cell (pepsinogen secretion), Gastric gland oxyntic cell (HCl secretion), Pancreatic acinar cell (bicarbonate and digestive enzyme secretion), Paneth cell of small intestine (lysozyme secretion), Type II pneumocyte of lung (surfactant secretion), Clara cell of lung, Hormone Secreting Cells, Anterior pituitary cell secreting growth hormone, Anterior pituitary cell secreting follicle- stimulating hormone, Anterior pituitary cell secreting luteinizing hormone, Anterior pituitary cell secreting prolactin, Anterior pituitary cell secreting adrenocorticotropic hormone, Anterior pituitary cell secreting thyroid-stimulating hormone, Intermediate pituitary cell secreting melanocyte-stimulating hormone, Posterior pituitary cell secreting oxytocin, Posterior pituitary cell secreting vasopressin, Gut and respiratory tract cell secreting serotonin, Gut and respiratory tract cell secreting endorphin, Gut and respiratory tract cell secreting somatostatin, Gut and respiratory tract cell secreting gastrin, Gut and respiratory tract cell secreting secretin, Gut and respiratory tract cell secreting cholecystokinin, Gut and respiratory tract cell secreting insulin, Gut and respiratory tract cell secreting glucagon, Gut and respiratory tract cell secreting bombesin, Thyroid gland cell secreting thyroid hormone, Thyroid gland cell secreting calcitonin, Parathyroid gland cell secreting parathyroid hormone, Parathyroid gland oxyphil cell, Adrenal gland cell secreting epinephrine, Adrenal gland cell secreting norepinephrine, Adrenal gland cell secreting steroid hormones (mineralcorticoids and gluco corticoids), Leydig cell of testes secreting testosterone, Theca interna cell of ovarian follicle secreting estrogen, Corpus luteum cell of ruptured ovarian follicle secreting progesterone, Kidney juxtaglomerular apparatus cell (renin secretion), Macula densa cell of kidney, Peripolar cell of kidney, Mesangial cell of kidney, Epithelial Absorptive Cells (Gut, Exocrine Glands and Urogenital Tract), Intestinal brush border cell (with microvilli), Exocrine gland striated duct cell, Gall bladder epithelial cell, Kidney proximal tubule brush border cell, Kidney distal tubule cell, Ductulus efferens nonciliated cell, Epididymal principal cell, Epididymal basal cell, Metabolism and Storage Cells, Hepatocyte (liver cell), White fat cell, Brown fat cell, Liver lipocyte, Barrier Function Cells (Lung, Gut, Exocrine Glands and Urogenital Tract), Type I pneumocyte (lining air space of lung), Pancreatic duct cell (centroacinar cell), Nonstriated duct cell (of sweat gland, salivary gland, mammary gland, etc.), Kidney glomerulus parietal cell, Kidney glomerulus podocyte, Loop of Henle thin segment cell (in kidney), Kidney collecting duct cell, Duct cell (of seminal vesicle, prostate gland, etc.), Epithelial Cells Lining Closed Internal Body Cavities, Blood vessel and lymphatic vascular endothelial fenestrated cell, Blood vessel and lymphatic vascular endothelial continuous cell, Blood vessel and lymphatic vascular endothelial splenic cell, Synovial cell (lining joint cavities, hyaluronic acid secretion), Serosal cell (lining peritoneal, pleural, and pericardial cavities), Squamous cell (lining perilymphatic space of ear), Squamous cell (lining endolymphatic space of ear), Columnar cell of endolymphatic sac with microvilli (lining endolymphatic space of ear), Columnar cell of endolymphatic sac without microvilli (lining endolymphatic space of ear), Dark cell (lining endolymphatic space of ear), Vestibular membrane cell (lining endolymphatic space of ear), Stria vascularis basal cell (lining endolymphatic space of ear), Stria vascularis marginal cell (lining endolymphatic space of ear), Cell of Claudius (lining endolymphatic space of ear), Cell of Boettcher (lining endolymphatic space of ear), Choroid plexus cell (cerebrospinal fluid secretion), Pia-arachnoid squamous cell, Pigmented ciliary epithelium cell of eye, Nonpigmented ciliary epithelium cell of eye, Corneal endothelial cell, Ciliated Cells with Propulsive Function, Respiratory tract ciliated cell, Oviduct ciliated cell (in female), Uterine endometrial ciliated cell (in female), Rete testis cilated cell (in male), Ductulus efferens ciliated cell (in male), Ciliated ependymal cell of central nervous system (lining brain cavities), Extracellular Matrix Secretion Cells, Ameloblast epithelial cell (tooth enamel secretion), Planum semilunatum epithelial cell of vestibular apparatus of ear (proteoglycan secretion), Organ of Corti interdental epithelial cell (secreting tectorial membrane covering hair cells), Loose connective tissue fibroblasts, Corneal fibroblasts, Tendon fibroblasts, Bone marrow reticular tissue fibroblasts, Other (nonepithelial) fibroblasts, Blood capillary pericyte, Nucleus pulposus cell of intervertebral disc, Cementoblast / cementocyte (tooth root bonelike cementum secretion), Odontoblast / odontocyte (tooth dentin secretion), Hyaline cartilage chondrocyte, Fibrocartilage chondrocyte, Elastic cartilage chondrocyte, Osteoblast / osteocyte, Osteoprogenitor cell (stem cell of osteoblasts), Hyalocyte of vitreous body of eye, Stellate cell of perilymphatic space of ear, Contractile Cells, Red skeletal muscle cell (slow), White skeletal muscle cell (fast), Intermediate skeletal muscle cell, Muscle spindle -- nuclear bag cell, Muscle spindle -- nuclear chain cell, Satellite cell (stem cell), Ordinary heart muscle cell, Nodal heart muscle cell, Purkinje fiber cell, Smooth muscle cell (various types), Myoepithelial cell of iris, Myoepithelial cell of exocrine glands, Blood and Immune System Cells, Erythrocyte (red blood cell), Megakaryocyte, Monocyte, Connective tissue macrophage (various types), Epidermal Langerhans cell, Osteoclast (in bone), Dendritic cell (in lymphoid tissues), Microglial cell (in central nervous system), Neutrophil, Eosinophil, Basophil, Mast cell, Helper T lymphocyte cell, Suppressor T lymphocyte cell, Killer T lymphocyte cell, IgM B lymphocyte cell, IgG B lymphocyte cell, IgA B lymphocyte cell, IgE B lymphocyte cell, Killer cell, Stem cells and committed progenitors for the blood and immune system (various types), Sensory Transducer Cells, Photoreceptor rod cell of eye, Photoreceptor blue-sensitive cone cell of eye, Photoreceptor green-sensitive cone cell of eye, Photoreceptor red-sensitive cone cell of eye, Auditory inner hair cell of organ of Corti, Auditory outer hair cell of organ of Corti, Type I hair cell of vestibular apparatus of ear (acceleration and gravity), Type II hair cell of vestibular apparatus of ear (acceleration and gravity), Type I taste bud cell, Olfactory neuron, Basal cell of olfactory epithelium (stem cell for olfactory neurons), Type I carotid body cell (blood pH sensor), Type II carotid body cell (blood pH sensor), Merkel cell of epidermis (touch sensor), Touch-sensitive 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 Neuron Cells, Cholinergic neural cell (various types), Adrenergic neural cell (various types), Peptidergic neural cell (various types), Sense Organ and Peripheral Neuron Supporting Cells, Inner pillar cell of organ of Corti, Outer pillar cell of organ of Corti, Inner phalangeal cell of organ of Corti, Outer phalangeal cell of organ of Corti, Border cell of organ of Corti, Hensen cell of organ of Corti, Vestibular apparatus supporting cell, Type I taste bud supporting cell, Olfactory epithelium supporting cell, Schwann cell, Satellite cell (encapsulating peripheral nerve cell bodies), Enteric glial cell, Central Nervous System Neurons and Glial Cells, Neuron cell (large variety of types, still poorly classified), Astrocyte glial cell (various types), Oligodendrocyte glial cell, Lens Cells, Anterior lens epithelial cell, Crystallin-containing lens fiber cell, Pigment Cells, Melanocyte, Retinal pigmented epithelial cell, Germ Cells, Oogonium / oocyte, Spermatocyte, Spermatogonium cell (stem cell for spermatocyte), Nurse Cells, Ovarian follicle cell, Sertoli cell (in testis), and Thymus epithelial cell. In some cases, the cells are mesenchymal stem cells (MSCs) or bone marrow stromal cells (BMSCs). These terms are used synonymously throughout herein. MSCs are of interest because they are easily isolated from a small aspirate of bone marrow, or other mesenchymal stem cell sources, and they readily generate single-cell derived colonies. Bone marrow cells may be obtained from iliac crest, femora, tibiae, spine, rib, knee or other mesenchymal tissues. Other sources of MSCs include embryonic yolk sac, placenta, umbilical cord, skin, fat, synovial tissue from joints, and blood. The presence of MSCs in culture colonies may be verified by specific cell surface markers which are identified with monoclonal antibodies. See U.S. Pat. Nos. 5,486,359 and 7,153,500. The single-cell derived colonies can be expanded through as many as 50 population doublings in about 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 Muscle Nerve 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 3001-3005; Woodbury et al., 2000 Neuroscience Res.61:364-370). In rare instances, the cells can differentiate into cells of all three germlines. Thus, MSCs serve as progenitors for multiple mesenchymal cell lineages including bone, cartilage, ligament, tendon, adipose, muscle, cardiac tissue, stroma, dermis, and other connective tissues. See U.S. Pat. Nos.6,387,369 and 7,101,704. For these reasons, MSCs currently are being tested for their potential use in cell and gene therapy of a number of human diseases (Horwitz et al., 1999 Nat. Med. 5:309-313; Caplan, et al. 2000 Clin. Orthoped.379:567-570). In some cases, MSCs can be defined by a variety of markers. For example, MSCs can be positive for CD73, CD90, CD166 and negative for CD14, CD34 and CD45. The cells can be derived from a human or other animal. For example, cells can originate from a mouse, guinea pig, rat, cattle, horses, pigs, sheep, or goat. In some embodiments, the cells originate from non-human primates. In some cases, the cells are used as autologous or allogenic treatment. Hyaluronidase can be applied to the culture medium in a wide range of amounts and has been found to be effective from 0.001 U / mL to 500 U / mL (see Example 1). Specifically, contemplated herein is the use of hyaluronidase in cell culture at the rate of 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, 26, 26.1, 26.2, 26.3, 26.4, 26.5, 26.6, 26.7, 26.8, 26.9, 27, 27.1, 27.2, 27.3, 27.4, 27.5, 27.6, 27.7, 27.8, 27.9, 28, 28.1, 28.2, 28.3, 28.4, 28.5, 28.6, 28.7, 28.8, 28.9, 29, 29.1, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9, 30, 30.1, 30.2, 30.3, 30.4, 30.5, 30.6, 30.7, 30.8, 30.9, 31, 31.1, 31.2, 31.3, 31.4, 31.5, 31.6, 31.7, 31.8, 31.9, 32, 32.1, 32.2, 32.3, 32.4, 32.5, 32.6, 32.7, 32.8, 32.9, 33, 33.1, 33.2, 33.3, 33.4, 33.5, 33.6, 33.7, 33.8, 33.9, 34, 34.1, 34.2, 34.3, 34.4, 34.5, 34.6, 34.7, 34.8, 34.9, 35, 35.1, 35.2, 35.3, 35.4, 35.5, 35.6, 35.7, 35.8, 35.9, 36, 36.1, 36.2, 36.3, 36.4, 36.5, 36.6, 36.7, 36.8, 36.9, 37, 37.1, 37.2, 37.3, 37.4, 37.5, 37.6, 37.7, 37.8, 37.9, 38, 38.1, 38.2, 38.3, 38.4, 38.5, 38.6, 38.7, 38.8, 38.9, 39, 39.1, 39.2, 39.3, 39.4, 39.5, 39.6, 39.7, 39.8, 39.9, 40, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9, 41, 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, 41.7, 41.8, 41.9, 42, 42.1, 42.2, 42.3, 42.4, 42.5, 42.6, 42.7, 42.8, 42.9, 43, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46, 46.1, 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48, 48.1, 48.2, 48.3, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, 49, 49.1, 49.2, 49.3, 49.4, 49.5, 49.6, 49.7, 49.8, 49.9, 50, 50.1, 50.2, 50.3, 50.4, 50.5, 50.6, 50.7, 50.8, 50.9, 51, 51.1, 51.2, 51.3, 51.4, 51.5, 51.6, 51.7, 51.8, 51.9, 52, 52.1, 52.2, 52.3, 52.4, 52.5, 52.6, 52.7, 52.8, 52.9, 53, 53.1, 53.2, 53.3, 53.4, 53.5, 53.6, 53.7, 53.8, 53.9, 54, 54.1, 54.2, 54.3, 54.4, 54.5, 54.6, 54.7, 54.8, 54.9, 55, 55.1, 55.2, 55.3, 55.4, 55.5, 55.6, 55.7, 55.8, 55.9, 56, 56.1, 56.2, 56.3, 56.4, 56.5, 56.6, 56.7, 56.8, 56.9, 57, 57.1, 57.2, 57.3, 57.4, 57.5, 57.6, 57.7, 57.8, 57.9, 58, 58.1, 58.2, 58.3, 58.4, 58.5, 58.6, 58.7, 58.8, 58.9, 59, 59.1, 59.2, 59.3, 59.4, 59.5, 59.6, 59.7, 59.8, 59.9, 60, 60.1, 60.2, 60.3, 60.4, 60.5, 60.6, 60.7, 60.8, 60.9, 61, 61.1, 61.2, 61.3, 61.4, 61.5, 61.6, 61.7, 61.8, 61.9, 62, 62.1, 62.2, 62.3, 62.4, 62.5, 62.6, 62.7, 62.8, 62.9, 63, 63.1, 63.2, 63.3, 63.4, 63.5, 63.6, 63.7, 63.8, 63.9, 64, 64.1, 64.2, 64.3, 64.4, 64.5, 64.6, 64.7, 64.8, 64.9, 65, 65.1, 65.2, 65.3, 65.4, 65.5, 65.6, 65.7, 65.8, 65.9, 66, 66.1, 66.2, 66.3, 66.4, 66.5, 66.6, 66.7, 66.8, 66.9, 67, 67.1, 67.2, 67.3, 67.4, 67.5, 67.6, 67.7, 67.8, 67.9, 68, 68.1, 68.2, 68.3, 68.4, 68.5, 68.6, 68.7, 68.8, 68.9, 69, 69.1, 69.2, 69.3, 69.4, 69.5, 69.6, 69.7, 69.8, 69.9, 70, 70.1, 70.2, 70.3, 70.4, 70.5, 70.6, 70.7, 70.8, 70.9, 71, 71.1, 71.2, 71.3, 71.4, 71.5, 71.6, 71.7, 71.8, 71.9, 72, 72.1, 72.2, 72.3, 72.4, 72.5, 72.6, 72.7, 72.8, 72.9, 73, 73.1, 73.2, 73.3, 73.4, 73.5, 73.6, 73.7, 73.8, 73.9, 74, 74.1, 74.2, 74.3, 74.4, 74.5, 74.6, 74.7, 74.8, 74.9, 75, 75.1, 75.2, 75.3, 75.4, 75.5, 75.6, 75.7, 75.8, 75.9, 76, 76.1, 76.2, 76.3, 76.4, 76.5, 76.6, 76.7, 76.8, 76.9, 77, 77.1, 77.2, 77.3, 77.4, 77.5, 77.6, 77.7, 77.8, 77.9, 78, 78.1, 78.2, 78.3, 78.4, 78.5, 78.6, 78.7, 78.8, 78.9, 79, 79.1, 79.2, 79.3, 79.4, 79.5, 79.6, 79.7, 79.8, 79.9, 80, 80.1, 80.2, 80.3, 80.4, 80.5, 80.6, 80.7, 80.8, 80.9, 81, 81.1, 81.2, 81.3, 81.4, 81.5, 81.6, 81.7, 81.8, 81.9, 82, 82.1, 82.2, 82.3, 82.4, 82.5, 82.6, 82.7, 82.8, 82.9, 83, 83.1, 83.2, 83.3, 83.4, 83.5, 83.6, 83.7, 83.8, 83.9, 84, 84.1, 84.2, 84.3, 84.4, 84.5, 84.6, 84.7, 84.8, 84.9, 85, 85.1, 85.2, 85.3, 85.4, 85.5, 85.6, 85.7, 85.8, 85.9, 86, 86.1, 86.2, 86.3, 86.4, 86.5, 86.6, 86.7, 86.8, 86.9, 87, 87.1, 87.2, 87.3, 87.4, 87.5, 87.6, 87.7, 87.8, 87.9, 88, 88.1, 88.2, 88.3, 88.4, 88.5, 88.6, 88.7, 88.8, 88.9, 89, 89.1, 89.2, 89.3, 89.4, 89.5, 89.6, 89.7, 89.8, 89.9, 90, 90.1, 90.2, 90.3, 90.4, 90.5, 90.6, 90.7, 90.8, 90.9, 91, 91.1, 91.2, 91.3, 91.4, 91.5, 91.6, 91.7, 91.8, 91.9, 92, 92.1, 92.2, 92.3, 92.4, 92.5, 92.6, 92.7, 92.8, 92.9, 93, 93.1, 93.2, 93.3, 93.4, 93.5, 93.6, 93.7, 93.8, 93.9, 94, 94.1, 94.2, 94.3, 94.4, 94.5, 94.6, 94.7, 94.8, 94.9, 95, 95.1, 95.2, 95.3, 95.4, 95.5, 95.6, 95.7, 95.8, 95.9, 96, 96.1, 96.2, 96.3, 96.4, 96.5, 96.6, 96.7, 96.8, 96.9, 97, 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.9, 98, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, 100, 100.1, 100.2, 100.3, 100.4, 100.5, 100.6, 100.7, 100.8, 100.9, 101, 101.1, 101.2, 101.3, 101.4, 101.5, 101.6, 101.7, 101.8, 101.9, 102, 102.1, 102.2, 102.3, 102.4, 102.5, 102.6, 102.7, 102.8, 102.9, 103, 103.1, 103.2, 103.3, 103.4, 103.5, 103.6, 103.7, 103.8, 103.9, 104, 104.1, 104.2, 104.3, 104.4, 104.5, 104.6, 104.7, 104.8, 104.9, 105, 105.1, 105.2, 105.3, 105.4, 105.5, 105.6, 105.7, 105.8, 105.9, 106, 106.1, 106.2, 106.3, 106.4, 106.5, 106.6, 106.7, 106.8, 106.9, 107, 107.1, 107.2, 107.3, 107.4, 107.5, 107.6, 107.7, 107.8, 107.9, 108, 108.1, 108.2, 108.3, 108.4, 108.5, 108.6, 108.7, 108.8, 108.9, 109, 109.1, 109.2, 109.3, 109.4, 109.5, 109.6, 109.7, 109.8, 109.9, 110, 110.1, 110.2, 110.3, 110.4, 110.5, 110.6, 110.7, 110.8, 110.9, 111, 111.1, 111.2, 111.3, 111.4, 111.5, 111.6, 111.7, 111.8, 111.9, 112, 112.1, 112.2, 112.3, 112.4, 112.5, 112.6, 112.7, 112.8, 112.9, 113, 113.1, 113.2, 113.3, 113.4, 113.5, 113.6, 113.7, 113.8, 113.9, 114, 114.1, 114.2, 114.3, 114.4, 114.5, 114.6, 114.7, 114.8, 114.9, 115, 115.1, 115.2, 115.3, 115.4, 115.5, 115.6, 115.7, 115.8, 115.9, 116, 116.1, 116.2, 116.3, 116.4, 116.5, 116.6, 116.7, 116.8, 116.9, 117, 117.1, 117.2, 117.3, 117.4, 117.5, 117.6, 117.7, 117.8, 117.9, 118, 118.1, 118.2, 118.3, 118.4, 118.5, 118.6, 118.7, 118.8, 118.9, 119, 119.1, 119.2, 119.3, 119.4, 119.5, 119.6, 119.7, 119.8, 119.9, 120, 120.1, 120.2, 120.3, 120.4, 120.5, 120.6, 120.7, 120.8, 120.9, 121, 121.1, 121.2, 121.3, 121.4, 121.5, 121.6, 121.7, 121.8, 121.9, 122, 122.1, 122.2, 122.3, 122.4, 122.5, 122.6, 122.7, 122.8, 122.9, 123, 123.1, 123.2, 123.3, 123.4, 123.5, 123.6, 123.7, 123.8, 123.9, 124, 124.1, 124.2, 124.3, 124.4, 124.5, 124.6, 124.7, 124.8, 124.9, 125, 125.1, 125.2, 125.3, 125.4, 125.5, 125.6, 125.7, 125.8, 125.9, 126, 126.1, 126.2, 126.3, 126.4, 126.5, 126.6, 126.7, 126.8, 126.9, 127, 127.1, 127.2, 127.3, 127.4, 127.5, 127.6, 127.7, 127.8, 127.9, 128, 128.1, 128.2, 128.3, 128.4, 128.5, 128.6, 128.7, 128.8, 128.9, 129, 129.1, 129.2, 129.3, 129.4, 129.5, 129.6, 129.7, 129.8, 129.9, 130, 130.1, 130.2, 130.3, 130.4, 130.5, 130.6, 130.7, 130.8, 130.9, 131, 131.1, 131.2, 131.3, 131.4, 131.5, 131.6, 131.7, 131.8, 131.9, 132, 132.1, 132.2, 132.3, 132.4, 132.5, 132.6, 132.7, 132.8, 132.9, 133, 133.1, 133.2, 133.3, 133.4, 133.5, 133.6, 133.7, 133.8, 133.9, 134, 134.1, 134.2, 134.3, 134.4, 134.5, 134.6, 134.7, 134.8, 134.9, 135, 135.1, 135.2, 135.3, 135.4, 135.5, 135.6, 135.7, 135.8, 135.9, 136, 136.1, 136.2, 136.3, 136.4, 136.5, 136.6, 136.7, 136.8, 136.9, 137, 137.1, 137.2, 137.3, 137.4, 137.5, 137.6, 137.7, 137.8, 137.9, 138, 138.1, 138.2, 138.3, 138.4, 138.5, 138.6, 138.7, 138.8, 138.9, 139, 139.1, 139.2, 139.3, 139.4, 139.5, 139.6, 139.7, 139.8, 139.9, 140, 140.1, 140.2, 140.3, 140.4, 140.5, 140.6, 140.7, 140.8, 140.9, 141, 141.1, 141.2, 141.3, 141.4, 141.5, 141.6, 141.7, 141.8, 141.9, 142, 142.1, 142.2, 142.3, 142.4, 142.5, 142.6, 142.7, 142.8, 142.9, 143, 143.1, 143.2, 143.3, 143.4, 143.5, 143.6, 143.7, 143.8, 143.9, 144, 144.1, 144.2, 144.3, 144.4, 144.5, 144.6, 144.7, 144.8, 144.9, 145, 145.1, 145.2, 145.3, 145.4, 145.5, 145.6, 145.7, 145.8, 145.9, 146, 146.1, 146.2, 146.3, 146.4, 146.5, 146.6, 146.7, 146.8, 146.9, 147, 147.1, 147.2, 147.3, 147.4, 147.5, 147.6, 147.7, 147.8, 147.9, 148, 148.1, 148.2, 148.3, 148.4, 148.5, 148.6, 148.7, 148.8, 148.9, 149, 149.1, 149.2, 149.3, 149.4, 149.5, 149.6, 149.7, 149.8, 149.9, 150, 150.1, 150.2, 150.3, 150.4, 150.5, 150.6, 150.7, 150.8, 150.9, 151, 151.1, 151.2, 151.3, 151.4, 151.5, 151.6, 151.7, 151.8, 151.9, 152, 152.1, 152.2, 152.3, 152.4, 152.5, 152.6, 152.7, 152.8, 152.9, 153, 153.1, 153.2, 153.3, 153.4, 153.5, 153.6, 153.7, 153.8, 153.9, 154, 154.1, 154.2, 154.3, 154.4, 154.5, 154.6, 154.7, 154.8, 154.9, 155, 155.1, 155.2, 155.3, 155.4, 155.5, 155.6, 155.7, 155.8, 155.9, 156, 156.1, 156.2, 156.3, 156.4, 156.5, 156.6, 156.7, 156.8, 156.9, 157, 157.1, 157.2, 157.3, 157.4, 157.5, 157.6, 157.7, 157.8, 157.9, 158, 158.1, 158.2, 158.3, 158.4, 158.5, 158.6, 158.7, 158.8, 158.9, 159, 159.1, 159.2, 159.3, 159.4, 159.5, 159.6, 159.7, 159.8, 159.9, 160, 160.1, 160.2, 160.3, 160.4, 160.5, 160.6, 160.7, 160.8, 160.9, 161, 161.1, 161.2, 161.3, 161.4, 161.5, 161.6, 161.7, 161.8, 161.9, 162, 162.1, 162.2, 162.3, 162.4, 162.5, 162.6, 162.7, 162.8, 162.9, 163, 163.1, 163.2, 163.3, 163.4, 163.5, 163.6, 163.7, 163.8, 163.9, 164, 164.1, 164.2, 164.3, 164.4, 164.5, 164.6, 164.7, 164.8, 164.9, 165, 165.1, 165.2, 165.3, 165.4, 165.5, 165.6, 165.7, 165.8, 165.9, 166, 166.1, 166.2, 166.3, 166.4, 166.5, 166.6, 166.7, 166.8, 166.9, 167, 167.1, 167.2, 167.3, 167.4, 167.5, 167.6, 167.7, 167.8, 167.9, 168, 168.1, 168.2, 168.3, 168.4, 168.5, 168.6, 168.7, 168.8, 168.9, 169, 169.1, 169.2, 169.3, 169.4, 169.5, 169.6, 169.7, 169.8, 169.9, 170, 170.1, 170.2, 170.3, 170.4, 170.5, 170.6, 170.7, 170.8, 170.9, 171, 171.1, 171.2, 171.3, 171.4, 171.5, 171.6, 171.7, 171.8, 171.9, 172, 172.1, 172.2, 172.3, 172.4, 172.5, 172.6, 172.7, 172.8, 172.9, 173, 173.1, 173.2, 173.3, 173.4, 173.5, 173.6, 173.7, 173.8, 173.9, 174, 174.1, 174.2, 174.3, 174.4, 174.5, 174.6, 174.7, 174.8, 174.9, 175, 175.1, 175.2, 175.3, 175.4, 175.5, 175.6, 175.7, 175.8, 175.9, 176, 176.1, 176.2, 176.3, 176.4, 176.5, 176.6, 176.7, 176.8, 176.9, 177, 177.1, 177.2, 177.3, 177.4, 177.5, 177.6, 177.7, 177.8, 177.9, 178, 178.1, 178.2, 178.3, 178.4, 178.5, 178.6, 178.7, 178.8, 178.9, 179, 179.1, 179.2, 179.3, 179.4, 179.5, 179.6, 179.7, 179.8, 179.9, 180, 180.1, 180.2, 180.3, 180.4, 180.5, 180.6, 180.7, 180.8, 180.9, 181, 181.1, 181.2, 181.3, 181.4, 181.5, 181.6, 181.7, 181.8, 181.9, 182, 182.1, 182.2, 182.3, 182.4, 182.5, 182.6, 182.7, 182.8, 182.9, 183, 183.1, 183.2, 183.3, 183.4, 183.5, 183.6, 183.7, 183.8, 183.9, 184, 184.1, 184.2, 184.3, 184.4, 184.5, 184.6, 184.7, 184.8, 184.9, 185, 185.1, 185.2, 185.3, 185.4, 185.5, 185.6, 185.7, 185.8, 185.9, 186, 186.1, 186.2, 186.3, 186.4, 186.5, 186.6, 186.7, 186.8, 186.9, 187, 187.1, 187.2, 187.3, 187.4, 187.5, 187.6, 187.7, 187.8, 187.9, 188, 188.1, 188.2, 188.3, 188.4, 188.5, 188.6, 188.7, 188.8, 188.9, 189, 189.1, 189.2, 189.3, 189.4, 189.5, 189.6, 189.7, 189.8, 189.9, 190, 190.1, 190.2, 190.3, 190.4, 190.5, 190.6, 190.7, 190.8, 190.9, 191, 191.1, 191.2, 191.3, 191.4, 191.5, 191.6, 191.7, 191.8, 191.9, 192, 192.1, 192.2, 192.3, 192.4, 192.5, 192.6, 192.7, 192.8, 192.9, 193, 193.1, 193.2, 193.3, 193.4, 193.5, 193.6, 193.7, 193.8, 193.9, 194, 194.1, 194.2, 194.3, 194.4, 194.5, 194.6, 194.7, 194.8, 194.9, 195, 195.1, 195.2, 195.3, 195.4, 195.5, 195.6, 195.7, 195.8, 195.9, 196, 196.1, 196.2, 196.3, 196.4, 196.5, 196.6, 196.7, 196.8, 196.9, 197, 197.1, 197.2, 197.3, 197.4, 197.5, 197.6, 197.7, 197.8, 197.9, 198, 198.1, 198.2, 198.3, 198.4, 198.5, 198.6, 198.7, 198.8, 198.9, 199, 199.1, 199.2, 199.3, 199.4, 199.5, 199.6, 199.7, 199.8, 199.9, 200, 200.1, 200.2, 200.3, 200.4, 200.5, 200.6, 200.7, 200.8, 200.9, 201, 201.1, 201.2, 201.3, 201.4, 201.5, 201.6, 201.7, 201.8, 201.9, 202, 202.1, 202.2, 202.3, 202.4, 202.5, 202.6, 202.7, 202.8, 202.9, 203, 203.1, 203.2, 203.3, 203.4, 203.5, 203.6, 203.7, 203.8, 203.9, 204, 204.1, 204.2, 204.3, 204.4, 204.5, 204.6, 204.7, 204.8, 204.9, 205, 205.1, 205.2, 205.3, 205.4, 205.5, 205.6, 205.7, 205.8, 205.9, 206, 206.1, 206.2, 206.3, 206.4, 206.5, 206.6, 206.7, 206.8, 206.9, 207, 207.1, 207.2, 207.3, 207.4, 207.5, 207.6, 207.7, 207.8, 207.9, 208, 208.1, 208.2, 208.3, 208.4, 208.5, 208.6, 208.7, 208.8, 208.9, 209, 209.1, 209.2, 209.3, 209.4, 209.5, 209.6, 209.7, 209.8, 209.9, 210, 210.1, 210.2, 210.3, 210.4, 210.5, 210.6, 210.7, 210.8, 210.9, 211, 211.1, 211.2, 211.3, 211.4, 211.5, 211.6, 211.7, 211.8, 211.9, 212, 212.1, 212.2, 212.3, 212.4, 212.5, 212.6, 212.7, 212.8, 212.9, 213, 213.1, 213.2, 213.3, 213.4, 213.5, 213.6, 213.7, 213.8, 213.9, 214, 214.1, 214.2, 214.3, 214.4, 214.5, 214.6, 214.7, 214.8, 214.9, 215, 215.1, 215.2, 215.3, 215.4, 215.5, 215.6, 215.7, 215.8, 215.9, 216, 216.1, 216.2, 216.3, 216.4, 216.5, 216.6, 216.7, 216.8, 216.9, 217, 217.1, 217.2, 217.3, 217.4, 217.5, 217.6, 217.7, 217.8, 217.9, 218, 218.1, 218.2, 218.3, 218.4, 218.5, 218.6, 218.7, 218.8, 218.9, 219, 219.1, 219.2, 219.3, 219.4, 219.5, 219.6, 219.7, 219.8, 219.9, 220, 220.1, 220.2, 220.3, 220.4, 220.5, 220.6, 220.7, 220.8, 220.9, 221, 221.1, 221.2, 221.3, 221.4, 221.5, 221.6, 221.7, 221.8, 221.9, 222, 222.1, 222.2, 222.3, 222.4, 222.5, 222.6, 222.7, 222.8, 222.9, 223, 223.1, 223.2, 223.3, 223.4, 223.5, 223.6, 223.7, 223.8, 223.9, 224, 224.1, 224.2, 224.3, 224.4, 224.5, 224.6, 224.7, 224.8, 224.9, 225, 225.1, 225.2, 225.3, 225.4, 225.5, 225.6, 225.7, 225.8, 225.9, 226, 226.1, 226.2, 226.3, 226.4, 226.5, 226.6, 226.7, 226.8, 226.9, 227, 227.1, 227.2, 227.3, 227.4, 227.5, 227.6, 227.7, 227.8, 227.9, 228, 228.1, 228.2, 228.3, 228.4, 228.5, 228.6, 228.7, 228.8, 228.9, 229, 229.1, 229.2, 229.3, 229.4, 229.5, 229.6, 229.7, 229.8, 229.9, 230, 230.1, 230.2, 230.3, 230.4, 230.5, 230.6, 230.7, 230.8, 230.9, 231, 231.1, 231.2, 231.3, 231.4, 231.5, 231.6, 231.7, 231.8, 231.9, 232, 232.1, 232.2, 232.3, 232.4, 232.5, 232.6, 232.7, 232.8, 232.9, 233, 233.1, 233.2, 233.3, 233.4, 233.5, 233.6, 233.7, 233.8, 233.9, 234, 234.1, 234.2, 234.3, 234.4, 234.5, 234.6, 234.7, 234.8, 234.9, 235, 235.1, 235.2, 235.3, 235.4, 235.5, 235.6, 235.7, 235.8, 235.9, 236, 236.1, 236.2, 236.3, 236.4, 236.5, 236.6, 236.7, 236.8, 236.9, 237, 237.1, 237.2, 237.3, 237.4, 237.5, 237.6, 237.7, 237.8, 237.9, 238, 238.1, 238.2, 238.3, 238.4, 238.5, 238.6, 238.7, 238.8, 238.9, 239, 239.1, 239.2, 239.3, 239.4, 239.5, 239.6, 239.7, 239.8, 239.9, 240, 240.1, 240.2, 240.3, 240.4, 240.5, 240.6, 240.7, 240.8, 240.9, 241, 241.1, 241.2, 241.3, 241.4, 241.5, 241.6, 241.7, 241.8, 241.9, 242, 242.1, 242.2, 242.3, 242.4, 242.5, 242.6, 242.7, 242.8, 242.9, 243, 243.1, 243.2, 243.3, 243.4, 243.5, 243.6, 243.7, 243.8, 243.9, 244, 244.1, 244.2, 244.3, 244.4, 244.5, 244.6, 244.7, 244.8, 244.9, 245, 245.1, 245.2, 245.3, 245.4, 245.5, 245.6, 245.7, 245.8, 245.9, 246, 246.1, 246.2, 246.3, 246.4, 246.5, 246.6, 246.7, 246.8, 246.9, 247, 247.1, 247.2, 247.3, 247.4, 247.5, 247.6, 247.7, 247.8, 247.9, 248, 248.1, 248.2, 248.3, 248.4, 248.5, 248.6, 248.7, 248.8, 248.9, 249, 249.1, 249.2, 249.3, 249.4, 249.5, 249.6, 249.7, 249.8, 249.9, 250, 250.1, 250.2, 250.3, 250.4, 250.5, 250.6, 250.7, 250.8, 250.9, 251, 251.1, 251.2, 251.3, 251.4, 251.5, 251.6, 251.7, 251.8, 251.9, 252, 252.1, 252.2, 252.3, 252.4, 252.5, 252.6, 252.7, 252.8, 252.9, 253, 253.1, 253.2, 253.3, 253.4, 253.5, 253.6, 253.7, 253.8, 253.9, 254, 254.1, 254.2, 254.3, 254.4, 254.5, 254.6, 254.7, 254.8, 254.9, 255, 255.1, 255.2, 255.3, 255.4, 255.5, 255.6, 255.7, 255.8, 255.9, 256, 256.1, 256.2, 256.3, 256.4, 256.5, 256.6, 256.7, 256.8, 256.9, 257, 257.1, 257.2, 257.3, 257.4, 257.5, 257.6, 257.7, 257.8, 257.9, 258, 258.1, 258.2, 258.3, 258.4, 258.5, 258.6, 258.7, 258.8, 258.9, 259, 259.1, 259.2, 259.3, 259.4, 259.5, 259.6, 259.7, 259.8, 259.9, 260, 260.1, 260.2, 260.3, 260.4, 260.5, 260.6, 260.7, 260.8, 260.9, 261, 261.1, 261.2, 261.3, 261.4, 261.5, 261.6, 261.7, 261.8, 261.9, 262, 262.1, 262.2, 262.3, 262.4, 262.5, 262.6, 262.7, 262.8, 262.9, 263, 263.1, 263.2, 263.3, 263.4, 263.5, 263.6, 263.7, 263.8, 263.9, 264, 264.1, 264.2, 264.3, 264.4, 264.5, 264.6, 264.7, 264.8, 264.9, 265, 265.1, 265.2, 265.3, 265.4, 265.5, 265.6, 265.7, 265.8, 265.9, 266, 266.1, 266.2, 266.3, 266.4, 266.5, 266.6, 266.7, 266.8, 266.9, 267, 267.1, 267.2, 267.3, 267.4, 267.5, 267.6, 267.7, 267.8, 267.9, 268, 268.1, 268.2, 268.3, 268.4, 268.5, 268.6, 268.7, 268.8, 268.9, 269, 269.1, 269.2, 269.3, 269.4, 269.5, 269.6, 269.7, 269.8, 269.9, 270, 270.1, 270.2, 270.3, 270.4, 270.5, 270.6, 270.7, 270.8, 270.9, 271, 271.1, 271.2, 271.3, 271.4, 271.5, 271.6, 271.7, 271.8, 271.9, 272, 272.1, 272.2, 272.3, 272.4, 272.5, 272.6, 272.7, 272.8, 272.9, 273, 273.1, 273.2, 273.3, 273.4, 273.5, 273.6, 273.7, 273.8, 273.9, 274, 274.1, 274.2, 274.3, 274.4, 274.5, 274.6, 274.7, 274.8, 274.9, 275, 275.1, 275.2, 275.3, 275.4, 275.5, 275.6, 275.7, 275.8, 275.9, 276, 276.1, 276.2, 276.3, 276.4, 276.5, 276.6, 276.7, 276.8, 276.9, 277, 277.1, 277.2, 277.3, 277.4, 277.5, 277.6, 277.7, 277.8, 277.9, 278, 278.1, 278.2, 278.3, 278.4, 278.5, 278.6, 278.7, 278.8, 278.9, 279, 279.1, 279.2, 279.3, 279.4, 279.5, 279.6, 279.7, 279.8, 279.9, 280, 280.1, 280.2, 280.3, 280.4, 280.5, 280.6, 280.7, 280.8, 280.9, 281, 281.1, 281.2, 281.3, 281.4, 281.5, 281.6, 281.7, 281.8, 281.9, 282, 282.1, 282.2, 282.3, 282.4, 282.5, 282.6, 282.7, 282.8, 282.9, 283, 283.1, 283.2, 283.3, 283.4, 283.5, 283.6, 283.7, 283.8, 283.9, 284, 284.1, 284.2, 284.3, 284.4, 284.5, 284.6, 284.7, 284.8, 284.9, 285, 285.1, 285.2, 285.3, 285.4, 285.5, 285.6, 285.7, 285.8, 285.9, 286, 286.1, 286.2, 286.3, 286.4, 286.5, 286.6, 286.7, 286.8, 286.9, 287, 287.1, 287.2, 287.3, 287.4, 287.5, 287.6, 287.7, 287.8, 287.9, 288, 288.1, 288.2, 288.3, 288.4, 288.5, 288.6, 288.7, 288.8, 288.9, 289, 289.1, 289.2, 289.3, 289.4, 289.5, 289.6, 289.7, 289.8, 289.9, 290, 290.1, 290.2, 290.3, 290.4, 290.5, 290.6, 290.7, 290.8, 290.9, 291, 291.1, 291.2, 291.3, 291.4, 291.5, 291.6, 291.7, 291.8, 291.9, 292, 292.1, 292.2, 292.3, 292.4, 292.5, 292.6, 292.7, 292.8, 292.9, 293, 293.1, 293.2, 293.3, 293.4, 293.5, 293.6, 293.7, 293.8, 293.9, 294, 294.1, 294.2, 294.3, 294.4, 294.5, 294.6, 294.7, 294.8, 294.9, 295, 295.1, 295.2, 295.3, 295.4, 295.5, 295.6, 295.7, 295.8, 295.9, 296, 296.1, 296.2, 296.3, 296.4, 296.5, 296.6, 296.7, 296.8, 296.9, 297, 297.1, 297.2, 297.3, 297.4, 297.5, 297.6, 297.7, 297.8, 297.9, 298, 298.1, 298.2, 298.3, 298.4, 298.5, 298.6, 298.7, 298.8, 298.9, 299, 299.1, 299.2, 299.3, 299.4, 299.5, 299.6, 299.7, 299.8, 299.9, 300, 300.1, 300.2, 300.3, 300.4, 300.5, 300.6, 300.7, 300.8, 300.9, 301, 301.1, 301.2, 301.3, 301.4, 301.5, 301.6, 301.7, 301.8, 301.9, 302, 302.1, 302.2, 302.3, 302.4, 302.5, 302.6, 302.7, 302.8, 302.9, 303, 303.1, 303.2, 303.3, 303.4, 303.5, 303.6, 303.7, 303.8, 303.9, 304, 304.1, 304.2, 304.3, 304.4, 304.5, 304.6, 304.7, 304.8, 304.9, 305, 305.1, 305.2, 305.3, 305.4, 305.5, 305.6, 305.7, 305.8, 305.9, 306, 306.1, 306.2, 306.3, 306.4, 306.5, 306.6, 306.7, 306.8, 306.9, 307, 307.1, 307.2, 307.3, 307.4, 307.5, 307.6, 307.7, 307.8, 307.9, 308, 308.1, 308.2, 308.3, 308.4, 308.5, 308.6, 308.7, 308.8, 308.9, 309, 309.1, 309.2, 309.3, 309.4, 309.5, 309.6, 309.7, 309.8, 309.9, 310, 310.1, 310.2, 310.3, 310.4, 310.5, 310.6, 310.7, 310.8, 310.9, 311, 311.1, 311.2, 311.3, 311.4, 311.5, 311.6, 311.7, 311.8, 311.9, 312, 312.1, 312.2, 312.3, 312.4, 312.5, 312.6, 312.7, 312.8, 312.9, 313, 313.1, 313.2, 313.3, 313.4, 313.5, 313.6, 313.7, 313.8, 313.9, 314, 314.1, 314.2, 314.3, 314.4, 314.5, 314.6, 314.7, 314.8, 314.9, 315, 315.1, 315.2, 315.3, 315.4, 315.5, 315.6, 315.7, 315.8, 315.9, 316, 316.1, 316.2, 316.3, 316.4, 316.5, 316.6, 316.7, 316.8, 316.9, 317, 317.1, 317.2, 317.3, 317.4, 317.5, 317.6, 317.7, 317.8, 317.9, 318, 318.1, 318.2, 318.3, 318.4, 318.5, 318.6, 318.7, 318.8, 318.9, 319, 319.1, 319.2, 319.3, 319.4, 319.5, 319.6, 319.7, 319.8, 319.9, 320, 320.1, 320.2, 320.3, 320.4, 320.5, 320.6, 320.7, 320.8, 320.9, 321, 321.1, 321.2, 321.3, 321.4, 321.5, 321.6, 321.7, 321.8, 321.9, 322, 322.1, 322.2, 322.3, 322.4, 322.5, 322.6, 322.7, 322.8, 322.9, 323, 323.1, 323.2, 323.3, 323.4, 323.5, 323.6, 323.7, 323.8, 323.9, 324, 324.1, 324.2, 324.3, 324.4, 324.5, 324.6, 324.7, 324.8, 324.9, 325, 325.1, 325.2, 325.3, 325.4, 325.5, 325.6, 325.7, 325.8, 325.9, 326, 326.1, 326.2, 326.3, 326.4, 326.5, 326.6, 326.7, 326.8, 326.9, 327, 327.1, 327.2, 327.3, 327.4, 327.5, 327.6, 327.7, 327.8, 327.9, 328, 328.1, 328.2, 328.3, 328.4, 328.5, 328.6, 328.7, 328.8, 328.9, 329, 329.1, 329.2, 329.3, 329.4, 329.5, 329.6, 329.7, 329.8, 329.9, 330, 330.1, 330.2, 330.3, 330.4, 330.5, 330.6, 330.7, 330.8, 330.9, 331, 331.1, 331.2, 331.3, 331.4, 331.5, 331.6, 331.7, 331.8, 331.9, 332, 332.1, 332.2, 332.3, 332.4, 332.5, 332.6, 332.7, 332.8, 332.9, 333, 333.1, 333.2, 333.3, 333.4, 333.5, 333.6, 333.7, 333.8, 333.9, 334, 334.1, 334.2, 334.3, 334.4, 334.5, 334.6, 334.7, 334.8, 334.9, 335, 335.1, 335.2, 335.3, 335.4, 335.5, 335.6, 335.7, 335.8, 335.9, 336, 336.1, 336.2, 336.3, 336.4, 336.5, 336.6, 336.7, 336.8, 336.9, 337, 337.1, 337.2, 337.3, 337.4, 337.5, 337.6, 337.7, 337.8, 337.9, 338, 338.1, 338.2, 338.3, 338.4, 338.5, 338.6, 338.7, 338.8, 338.9, 339, 339.1, 339.2, 339.3, 339.4, 339.5, 339.6, 339.7, 339.8, 339.9, 340, 340.1, 340.2, 340.3, 340.4, 340.5, 340.6, 340.7, 340.8, 340.9, 341, 341.1, 341.2, 341.3, 341.4, 341.5, 341.6, 341.7, 341.8, 341.9, 342, 342.1, 342.2, 342.3, 342.4, 342.5, 342.6, 342.7, 342.8, 342.9, 343, 343.1, 343.2, 343.3, 343.4, 343.5, 343.6, 343.7, 343.8, 343.9, 344, 344.1, 344.2, 344.3, 344.4, 344.5, 344.6, 344.7, 344.8, 344.9, 345, 345.1, 345.2, 345.3, 345.4, 345.5, 345.6, 345.7, 345.8, 345.9, 346, 346.1, 346.2, 346.3, 346.4, 346.5, 346.6, 346.7, 346.8, 346.9, 347, 347.1, 347.2, 347.3, 347.4, 347.5, 347.6, 347.7, 347.8, 347.9, 348, 348.1, 348.2, 348.3, 348.4, 348.5, 348.6, 348.7, 348.8, 348.9, 349, 349.1, 349.2, 349.3, 349.4, 349.5, 349.6, 349.7, 349.8, 349.9, 350, 350.1, 350.2, 350.3, 350.4, 350.5, 350.6, 350.7, 350.8, 350.9, 351, 351.1, 351.2, 351.3, 351.4, 351.5, 351.6, 351.7, 351.8, 351.9, 352, 352.1, 352.2, 352.3, 352.4, 352.5, 352.6, 352.7, 352.8, 352.9, 353, 353.1, 353.2, 353.3, 353.4, 353.5, 353.6, 353.7, 353.8, 353.9, 354, 354.1, 354.2, 354.3, 354.4, 354.5, 354.6, 354.7, 354.8, 354.9, 355, 355.1, 355.2, 355.3, 355.4, 355.5, 355.6, 355.7, 355.8, 355.9, 356, 356.1, 356.2, 356.3, 356.4, 356.5, 356.6, 356.7, 356.8, 356.9, 357, 357.1, 357.2, 357.3, 357.4, 357.5, 357.6, 357.7, 357.8, 357.9, 358, 358.1, 358.2, 358.3, 358.4, 358.5, 358.6, 358.7, 358.8, 358.9, 359, 359.1, 359.2, 359.3, 359.4, 359.5, 359.6, 359.7, 359.8, 359.9, 360, 360.1, 360.2, 360.3, 360.4, 360.5, 360.6, 360.7, 360.8, 360.9, 361, 361.1, 361.2, 361.3, 361.4, 361.5, 361.6, 361.7, 361.8, 361.9, 362, 362.1, 362.2, 362.3, 362.4, 362.5, 362.6, 362.7, 362.8, 362.9, 363, 363.1, 363.2, 363.3, 363.4, 363.5, 363.6, 363.7, 363.8, 363.9, 364, 364.1, 364.2, 364.3, 364.4, 364.5, 364.6, 364.7, 364.8, 364.9, 365, 365.1, 365.2, 365.3, 365.4, 365.5, 365.6, 365.7, 365.8, 365.9, 366, 366.1, 366.2, 366.3, 366.4, 366.5, 366.6, 366.7, 366.8, 366.9, 367, 367.1, 367.2, 367.3, 367.4, 367.5, 367.6, 367.7, 367.8, 367.9, 368, 368.1, 368.2, 368.3, 368.4, 368.5, 368.6, 368.7, 368.8, 368.9, 369, 369.1, 369.2, 369.3, 369.4, 369.5, 369.6, 369.7, 369.8, 369.9, 370, 370.1, 370.2, 370.3, 370.4, 370.5, 370.6, 370.7, 370.8, 370.9, 371, 371.1, 371.2, 371.3, 371.4, 371.5, 371.6, 371.7, 371.8, 371.9, 372, 372.1, 372.2, 372.3, 372.4, 372.5, 372.6, 372.7, 372.8, 372.9, 373, 373.1, 373.2, 373.3, 373.4, 373.5, 373.6, 373.7, 373.8, 373.9, 374, 374.1, 374.2, 374.3, 374.4, 374.5, 374.6, 374.7, 374.8, 374.9, 375, 375.1, 375.2, 375.3, 375.4, 375.5, 375.6, 375.7, 375.8, 375.9, 376, 376.1, 376.2, 376.3, 376.4, 376.5, 376.6, 376.7, 376.8, 376.9, 377, 377.1, 377.2, 377.3, 377.4, 377.5, 377.6, 377.7, 377.8, 377.9, 378, 378.1, 378.2, 378.3, 378.4, 378.5, 378.6, 378.7, 378.8, 378.9, 379, 379.1, 379.2, 379.3, 379.4, 379.5, 379.6, 379.7, 379.8, 379.9, 380, 380.1, 380.2, 380.3, 380.4, 380.5, 380.6, 380.7, 380.8, 380.9, 381, 381.1, 381.2, 381.3, 381.4, 381.5, 381.6, 381.7, 381.8, 381.9, 382, 382.1, 382.2, 382.3, 382.4, 382.5, 382.6, 382.7, 382.8, 382.9, 383, 383.1, 383.2, 383.3, 383.4, 383.5, 383.6, 383.7, 383.8, 383.9, 384, 384.1, 384.2, 384.3, 384.4, 384.5, 384.6, 384.7, 384.8, 384.9, 385, 385.1, 385.2, 385.3, 385.4, 385.5, 385.6, 385.7, 385.8, 385.9, 386, 386.1, 386.2, 386.3, 386.4, 386.5, 386.6, 386.7, 386.8, 386.9, 387, 387.1, 387.2, 387.3, 387.4, 387.5, 387.6, 387.7, 387.8, 387.9, 388, 388.1, 388.2, 388.3, 388.4, 388.5, 388.6, 388.7, 388.8, 388.9, 389, 389.1, 389.2, 389.3, 389.4, 389.5, 389.6, 389.7, 389.8, 389.9, 390, 390.1, 390.2, 390.3, 390.4, 390.5, 390.6, 390.7, 390.8, 390.9, 391, 391.1, 391.2, 391.3, 391.4, 391.5, 391.6, 391.7, 391.8, 391.9, 392, 392.1, 392.2, 392.3, 392.4, 392.5, 392.6, 392.7, 392.8, 392.9, 393, 393.1, 393.2, 393.3, 393.4, 393.5, 393.6, 393.7, 393.8, 393.9, 394, 394.1, 394.2, 394.3, 394.4, 394.5, 394.6, 394.7, 394.8, 394.9, 395, 395.1, 395.2, 395.3, 395.4, 395.5, 395.6, 395.7, 395.8, 395.9, 396, 396.1, 396.2, 396.3, 396.4, 396.5, 396.6, 396.7, 396.8, 396.9, 397, 397.1, 397.2, 397.3, 397.4, 397.5, 397.6, 397.7, 397.8, 397.9, 398, 398.1, 398.2, 398.3, 398.4, 398.5, 398.6, 398.7, 398.8, 398.9, 399, 399.1, 399.2, 399.3, 399.4, 399.5, 399.6, 399.7, 399.8, 399.9, 400, 400.1, 400.2, 400.3, 400.4, 400.5, 400.6, 400.7, 400.8, 400.9, 401, 401.1, 401.2, 401.3, 401.4, 401.5, 401.6, 401.7, 401.8, 401.9, 402, 402.1, 402.2, 402.3, 402.4, 402.5, 402.6, 402.7, 402.8, 402.9, 403, 403.1, 403.2, 403.3, 403.4, 403.5, 403.6, 403.7, 403.8, 403.9, 404, 404.1, 404.2, 404.3, 404.4, 404.5, 404.6, 404.7, 404.8, 404.9, 405, 405.1, 405.2, 405.3, 405.4, 405.5, 405.6, 405.7, 405.8, 405.9, 406, 406.1, 406.2, 406.3, 406.4, 406.5, 406.6, 406.7, 406.8, 406.9, 407, 407.1, 407.2, 407.3, 407.4, 407.5, 407.6, 407.7, 407.8, 407.9, 408, 408.1, 408.2, 408.3, 408.4, 408.5, 408.6, 408.7, 408.8, 408.9, 409, 409.1, 409.2, 409.3, 409.4, 409.5, 409.6, 409.7, 409.8, 409.9, 410, 410.1, 410.2, 410.3, 410.4, 410.5, 410.6, 410.7, 410.8, 410.9, 411, 411.1, 411.2, 411.3, 411.4, 411.5, 411.6, 411.7, 411.8, 411.9, 412, 412.1, 412.2, 412.3, 412.4, 412.5, 412.6, 412.7, 412.8, 412.9, 413, 413.1, 413.2, 413.3, 413.4, 413.5, 413.6, 413.7, 413.8, 413.9, 414, 414.1, 414.2, 414.3, 414.4, 414.5, 414.6, 414.7, 414.8, 414.9, 415, 415.1, 415.2, 415.3, 415.4, 415.5, 415.6, 415.7, 415.8, 415.9, 416, 416.1, 416.2, 416.3, 416.4, 416.5, 416.6, 416.7, 416.8, 416.9, 417, 417.1, 417.2, 417.3, 417.4, 417.5, 417.6, 417.7, 417.8, 417.9, 418, 418.1, 418.2, 418.3, 418.4, 418.5, 418.6, 418.7, 418.8, 418.9, 419, 419.1, 419.2, 419.3, 419.4, 419.5, 419.6, 419.7, 419.8, 419.9, 420, 420.1, 420.2, 420.3, 420.4, 420.5, 420.6, 420.7, 420.8, 420.9, 421, 421.1, 421.2, 421.3, 421.4, 421.5, 421.6, 421.7, 421.8, 421.9, 422, 422.1, 422.2, 422.3, 422.4, 422.5, 422.6, 422.7, 422.8, 422.9, 423, 423.1, 423.2, 423.3, 423.4, 423.5, 423.6, 423.7, 423.8, 423.9, 424, 424.1, 424.2, 424.3, 424.4, 424.5, 424.6, 424.7, 424.8, 424.9, 425, 425.1, 425.2, 425.3, 425.4, 425.5, 425.6, 425.7, 425.8, 425.9, 426, 426.1, 426.2, 426.3, 426.4, 426.5, 426.6, 426.7, 426.8, 426.9, 427, 427.1, 427.2, 427.3, 427.4, 427.5, 427.6, 427.7, 427.8, 427.9, 428, 428.1, 428.2, 428.3, 428.4, 428.5, 428.6, 428.7, 428.8, 428.9, 429, 429.1, 429.2, 429.3, 429.4, 429.5, 429.6, 429.7, 429.8, 429.9, 430, 430.1, 430.2, 430.3, 430.4, 430.5, 430.6, 430.7, 430.8, 430.9, 431, 431.1, 431.2, 431.3, 431.4, 431.5, 431.6, 431.7, 431.8, 431.9, 432, 432.1, 432.2, 432.3, 432.4, 432.5, 432.6, 432.7, 432.8, 432.9, 433, 433.1, 433.2, 433.3, 433.4, 433.5, 433.6, 433.7, 433.8, 433.9, 434, 434.1, 434.2, 434.3, 434.4, 434.5, 434.6, 434.7, 434.8, 434.9, 435, 435.1, 435.2, 435.3, 435.4, 435.5, 435.6, 435.7, 435.8, 435.9, 436, 436.1, 436.2, 436.3, 436.4, 436.5, 436.6, 436.7, 436.8, 436.9, 437, 437.1, 437.2, 437.3, 437.4, 437.5, 437.6, 437.7, 437.8, 437.9, 438, 438.1, 438.2, 438.3, 438.4, 438.5, 438.6, 438.7, 438.8, 438.9, 439, 439.1, 439.2, 439.3, 439.4, 439.5, 439.6, 439.7, 439.8, 439.9, 440, 440.1, 440.2, 440.3, 440.4, 440.5, 440.6, 440.7, 440.8, 440.9, 441, 441.1, 441.2, 441.3, 441.4, 441.5, 441.6, 441.7, 441.8, 441.9, 442, 442.1, 442.2, 442.3, 442.4, 442.5, 442.6, 442.7, 442.8, 442.9, 443, 443.1, 443.2, 443.3, 443.4, 443.5, 443.6, 443.7, 443.8, 443.9, 444, 444.1, 444.2, 444.3, 444.4, 444.5, 444.6, 444.7, 444.8, 444.9, 445, 445.1, 445.2, 445.3, 445.4, 445.5, 445.6, 445.7, 445.8, 445.9, 446, 446.1, 446.2, 446.3, 446.4, 446.5, 446.6, 446.7, 446.8, 446.9, 447, 447.1, 447.2, 447.3, 447.4, 447.5, 447.6, 447.7, 447.8, 447.9, 448, 448.1, 448.2, 448.3, 448.4, 448.5, 448.6, 448.7, 448.8, 448.9, 449, 449.1, 449.2, 449.3, 449.4, 449.5, 449.6, 449.7, 449.8, 449.9, 450, 450.1, 450.2, 450.3, 450.4, 450.5, 450.6, 450.7, 450.8, 450.9, 451, 451.1, 451.2, 451.3, 451.4, 451.5, 451.6, 451.7, 451.8, 451.9, 452, 452.1, 452.2, 452.3, 452.4, 452.5, 452.6, 452.7, 452.8, 452.9, 453, 453.1, 453.2, 453.3, 453.4, 453.5, 453.6, 453.7, 453.8, 453.9, 454, 454.1, 454.2, 454.3, 454.4, 454.5, 454.6, 454.7, 454.8, 454.9, 455, 455.1, 455.2, 455.3, 455.4, 455.5, 455.6, 455.7, 455.8, 455.9, 456, 456.1, 456.2, 456.3, 456.4, 456.5, 456.6, 456.7, 456.8, 456.9, 457, 457.1, 457.2, 457.3, 457.4, 457.5, 457.6, 457.7, 457.8, 457.9, 458, 458.1, 458.2, 458.3, 458.4, 458.5, 458.6, 458.7, 458.8, 458.9, 459, 459.1, 459.2, 459.3, 459.4, 459.5, 459.6, 459.7, 459.8, 459.9, 460, 460.1, 460.2, 460.3, 460.4, 460.5, 460.6, 460.7, 460.8, 460.9, 461, 461.1, 461.2, 461.3, 461.4, 461.5, 461.6, 461.7, 461.8, 461.9, 462, 462.1, 462.2, 462.3, 462.4, 462.5, 462.6, 462.7, 462.8, 462.9, 463, 463.1, 463.2, 463.3, 463.4, 463.5, 463.6, 463.7, 463.8, 463.9, 464, 464.1, 464.2, 464.3, 464.4, 464.5, 464.6, 464.7, 464.8, 464.9, 465, 465.1, 465.2, 465.3, 465.4, 465.5, 465.6, 465.7, 465.8, 465.9, 466, 466.1, 466.2, 466.3, 466.4, 466.5, 466.6, 466.7, 466.8, 466.9, 467, 467.1, 467.2, 467.3, 467.4, 467.5, 467.6, 467.7, 467.8, 467.9, 468, 468.1, 468.2, 468.3, 468.4, 468.5, 468.6, 468.7, 468.8, 468.9, 469, 469.1, 469.2, 469.3, 469.4, 469.5, 469.6, 469.7, 469.8, 469.9, 470, 470.1, 470.2, 470.3, 470.4, 470.5, 470.6, 470.7, 470.8, 470.9, 471, 471.1, 471.2, 471.3, 471.4, 471.5, 471.6, 471.7, 471.8, 471.9, 472, 472.1, 472.2, 472.3, 472.4, 472.5, 472.6, 472.7, 472.8, 472.9, 473, 473.1, 473.2, 473.3, 473.4, 473.5, 473.6, 473.7, 473.8, 473.9, 474, 474.1, 474.2, 474.3, 474.4, 474.5, 474.6, 474.7, 474.8, 474.9, 475, 475.1, 475.2, 475.3, 475.4, 475.5, 475.6, 475.7, 475.8, 475.9, 476, 476.1, 476.2, 476.3, 476.4, 476.5, 476.6, 476.7, 476.8, 476.9, 477, 477.1, 477.2, 477.3, 477.4, 477.5, 477.6, 477.7, 477.8, 477.9, 478, 478.1, 478.2, 478.3, 478.4, 478.5, 478.6, 478.7, 478.8, 478.9, 479, 479.1, 479.2, 479.3, 479.4, 479.5, 479.6, 479.7, 479.8, 479.9, 480, 480.1, 480.2, 480.3, 480.4, 480.5, 480.6, 480.7, 480.8, 480.9, 481, 481.1, 481.2, 481.3, 481.4, 481.5, 481.6, 481.7, 481.8, 481.9, 482, 482.1, 482.2, 482.3, 482.4, 482.5, 482.6, 482.7, 482.8, 482.9, 483, 483.1, 483.2, 483.3, 483.4, 483.5, 483.6, 483.7, 483.8, 483.9, 484, 484.1, 484.2, 484.3, 484.4, 484.5, 484.6, 484.7, 484.8, 484.9, 485, 485.1, 485.2, 485.3, 485.4, 485.5, 485.6, 485.7, 485.8, 485.9, 486, 486.1, 486.2, 486.3, 486.4, 486.5, 486.6, 486.7, 486.8, 486.9, 487, 487.1, 487.2, 487.3, 487.4, 487.5, 487.6, 487.7, 487.8, 487.9, 488, 488.1, 488.2, 488.3, 488.4, 488.5, 488.6, 488.7, 488.8, 488.9, 489, 489.1, 489.2, 489.3, 489.4, 489.5, 489.6, 489.7, 489.8, 489.9, 490, 490.1, 490.2, 490.3, 490.4, 490.5, 490.6, 490.7, 490.8, 490.9, 491, 491.1, 491.2, 491.3, 491.4, 491.5, 491.6, 491.7, 491.8, 491.9, 492, 492.1, 492.2, 492.3, 492.4, 492.5, 492.6, 492.7, 492.8, 492.9, 493, 493.1, 493.2, 493.3, 493.4, 493.5, 493.6, 493.7, 493.8, 493.9, 494, 494.1, 494.2, 494.3, 494.4, 494.5, 494.6, 494.7, 494.8, 494.9, 495, 495.1, 495.2, 495.3, 495.4, 495.5, 495.6, 495.7, 495.8, 495.9, 496, 496.1, 496.2, 496.3, 496.4, 496.5, 496.6, 496.7, 496.8, 496.9, 497, 497.1, 497.2, 497.3, 497.4, 497.5, 497.6, 497.7, 497.8, 497.9, 498, 498.1, 498.2, 498.3, 498.4, 498.5, 498.6, 498.7, 498.8, 498.9, 499, 499.1, 499.2, 499.3, 499.4, 499.5, 499.6, 499.7, 499.8, 499.9, or 500 U / mL or any amount (concentration) above, below, or in-between these values. In a specific example, hyaluronidase is applied in a range between 0.001 U / ml to 10 U / ml. One of skill in the art can readily assess the rate to apply hyaluronidase based on the type of cell culture being used, the specific issues with viscosity which are being faced, and the desired viscosity of the culture. For example, it can be optimal to use hyaluronidase at 0.001 U / ml (or 1 mU / ml) when MSCs are cultured to a density of between 100,000 cells / mL and 1,000,000 cells / mL. When MSCs reach higher densities faster, the viscosity build quicker, which can be due to hyaluronic acid secretion by the cells into the culture medium. The buildup of HA over time leads to increasing medium viscosity, eventually leading to culture failure. The addition of hyaluronidase breaks down the HA and brings the viscosity down to levels that enable microcarriers to remain in suspension, and the cultures producing more EVs. If higher concentrations of hyaluronidase are added, such as 0.1 U / mL or higher, then the cells can fall off the microcarriers and no longer grow. Once the MSCs have produced EVs, be it in small scale flasks, or large scale bioreactors, then the medium is processed to purify the secretome which contains a variety of extracellular materials including EVs. Prior to TFF and chromatography, the conditioned medium must be clarified via depth filtration. This clarification step can begin with a large cutoff filter of several microns, and followed by smaller filters such as 0.8 micron and then 0.45 micron. This is typically begun by removing all large (~250 micron diameter) microcarriers from the conditioned medium with a 60-100 micron mesh. The remaining cells and cellular debris is removed often with a 5 micron filter, and then either a 0.8 or 0.45 micron filter to further clarify the medium prior to TFF. One significant challenge within EVs is that the conditioned medium rapidly clogs even filters as large as 5 microns, and while biological materials accumulates in the filter, creating a back pressure, much of the EVs get irreversibly locked in the filter, greatly reducing yields. Ti has been found that the addition of hyaluronidase to the conditioned medium enables a more streamlined filtration process with much less pressure build up, and surprisingly with a significantly increased amount of EVs getting through the filters. Several examples of increased yields of filters from 0.45 micron up to 5 micron is size are demonstrated herein. During culture hyaluronidase can be applied to the cell culture once, or at multiple times during the culture period depending on the viscosity build up rate. For example, hyaluronidase can be applied when the cell culture is established, or immediately before cells are harvested. It can be applied once a day, twice a day, three times a day, four times a day, or more. It can be applied every other day, or every third day, or every fourth day, or every fifth day, or every sixth day, or once a week, twice a week, three times a week, four times a week, five times a week, or six times a week. It can be applied every other week, every third week, every fourth week, or every month, or every other month, for example. It can be applied at regular intervals, or can be applied sporadically when needed. For example, one of skill 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 days 5 and 15, or between days 8 and 12. In that period it can be applied once, twice, three times, four times, five times, or more. Hyaluronidase can be added to cell culture at a variety of temperatures. For example, disclosed herein is the addition of hyaluronidase at 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, or 65°C, or any temperature above, below, or in-between these temperatures. Hyaluronidase can be added to the cell culture during conditions which are designed to improve yield of a desired product from the cell culture. For example, these co-optimized conditions include, but are not limited to, varied agitation rates and media compositions, wherein said addition can reduce viscosity and / or fouling of the cell culture quality and loss of cell suspension. By “varied agitation” is meant that agitation rates can vary from 20 RPM and up to 500 RPM, or a rate that is sufficient to maintain the cells and / or micro carriers in suspension without interfering with cell adhesion to microcarriers, or cell viability. By “media composition” is meant that cells, such as MSCs, can be grown my multiple media systems, ranging from DMEM + FBS, DMEM + hPL, or alphaMEM + FBS or hPL. Serum-free media is also contemplated herein. It is fully expected that when any of these media are used for extended culture of cells for EV production, asuch as MSCs, that hyaluronidase can aid in maintaining healthy suspension cultures, or aid in the filtration and purification of TVs from these media. Providing hyaluronidase to cell culture can greatly decrease viscosity of the same. For example, upon application, viscosity can be decreased 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%, 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 in- between these values. This value is compared to a cell culture control in which conditions remain identical except that hyaluronidase is not added to the control. Disclosed herein is a cell culture media comprising hyaluronidase. This cell culture media can be specific for MSCs, for example, and the cell culture media can include cells, as described above. The cell culture media can be RoosterCollect EV Pro™, RoosterCollect™ -EV, or EV Boost™. The cell culture media can comprise microcarriers, or can be concentrated, as described above. The hyaluronidase can be provided at a variety of concentrations, as described above. Also disclosed herein is a method for increasing yield of extracellular vesicles (EVs) during downstream processing. Conditioned media produced in the upstream process is a complex mixture that contains not only extracellular vesicles but also other biological components including cell debris, proteins, nucleic acids, lipids, and media components. Large- scale biomanufacturing of EVs for clinical applications involve a downstream process that concentrates, purifies, and formulates EV preparations to meet final product specifications without compromising their critical quality attributes (CQAs). The product 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 wellness, cosmetic, and / or theragnostic use. Specifically disclosed herein is a method of improving properties of liquid cell culture media, the method comprising culturing cells in the liquid medium, removing said cells, and then introducing HAase to the liquid medium after cells are removed. By doing this, the viscosity of the culture media can be significantly reduced. Reduction in viscosity is discussed above. A reduction in viscosity can lead to an increase in yield. This increase in yield compared to a control in which hyaluronidase is not used can be a 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%, 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% increase, or a 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more increase in yield. HAase can improve the yield, quality, or processing efficiency of one or more products secreted by the cells, including extracellular vesicles (EVs), exosomes, microvesicles, cellular ghosts, secretory granules, or virus particles, as discussed herein. Methods of using HAase during downstream processing are described in Example 3, below. The cells can be removed from liquid medium (or liquid medium can be removed from the cells), and then HAase can be added. Once this occurs, the liquid medium can be clarified and concentrated. Such clarification can take place by filtration. The filter can have a pore size of between 0.45um and 100 µm, for example. The liquid medium can then 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, 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 fold, or any amount below, above, or between these values. Amounts of HAase which can be added are discussed above and in Example 3. Specifically disclosed herein is a method comprising: producing extracellular vesicles within a bioreactor; and introducing hyaluronidase to the extracellular vesicles during downstream processing, thereby increasing yield of extracellular vesicles as compared to a control in which EVs are not exposed to hyaluronidase. The addition of hyaluronidase during downstream processing can occur at any point before filtration. For example, it can be added directly to the bioreactor prior to harvest, or it can be added after the cells and microcarriers are removed from the medium. It can be added before clarification takes place, 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 clarification steps. It can be added at more than one of these time points, and can be, for example, added at various points during downstream processing. Further disclosed herein is a composition comprising liquid cell culture media and exogenously added hyaluronidase. By “exogenously added” is meant that the HAase is not naturally produced by one of the cells within the culture, or is not produced in sufficient quantities to have the desired attributes, but is added separately to the liquid cell culture media. This liquid cell culture media may or may not comprise cells, such as MSCs. In some embodiments, the cells are attached to a microcarrier, wherein the microcarrier is in the liquid cell culture media. In a specific embodiment, cells of the composition can be at a density of 50,000 to 5,000,000 cells / mL, EVs can be within a concentration of 1E8 to 1E11 particles / mL, and exogenous hyaluronidase can be present at a concentration between 0.0001 U / mL and 100 U / mL. The liquid cell culture media of the composition can comprise one or more products secreted by the cells, including extracellular vesicles (EVs), exosomes, microvesicles, cellular ghosts, secretory granules, or virus particles. Also disclosed herein is a composition comprising conditioned medium derived from an MSC cell culture, wherein the conditioned media comprises EVs, wherein between 1E8 and 1E11 EVs are present, and further wherein the conditioned medium comprises exogenously added HAase at a concentration of about 0.01 to 100 U / mL, wherein the conditioned medium has not undergone clarification or filtration. Also disclosed is a kit comprising hyaluronidase and cell culture media. The hyaluronidase can be provided separately from the cell culture media, or can be within it. The kit can further comprise instructions for use. These instructions can include specific details regarding the amount, timing of application, or concentration to use of hyaluronidase. Other components can be provided within the kit as well. The hyaluronidase can be provided in amounts specified above. The cell culture media can be any of the types disclosed herein, and can be concentrated, or can comprise microcarriers. EXAMPLES Example 1: Compositions and Methods for Minimizing Viscosity of Culture Supernatant from Primary Cells MSC cell culture is performed with standardized expansion processes, with some variation of EV production that generates conditioned medium from which to purify EVs from. MSCs are sourced for these studies are commercially available from RoosterBio Inc as the RoosterVial product with MSCs sourced from human bone marrow, human umbilical cord, or human adipose tissue. The RoosterVial product cells are thawed, plated at 2000-3000 cells / cm2 in Corning CellBind flasks and expanded in RoosterNourish-XF, a xeno free bioprocess expansion medium, for 4-5 days or until 80-90% confluence. Alternative vendor medium is used at times and would be called out. After a single passage, cells are harvested using TrypLE (Thermo) and passaged into Corning CellBind flasks, or seeded into bioreactors (disposable Corning spinner flasks, or Sartorius Ambr 250ml vessels) at 23,000 cells / mL and expanded on microcarriers (Corning Synthemax or Sartorius Solohill Collagen-coated carriers) added at 42 mg / mL. These methods are published and can be found in (Lembong, J., Kirian, R., Takacs, J.D., Olsen, T.R., Lock, L.T., Rowley, J.A., 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, J.A., 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 are expanded for 5 days in RoosterNourish culture medium with the addition of a day 3 feed of RoosterReplenish. After 5 days the RoosterNourish expansion medium is removed, the cells on microcarriers are gentely washed and then an EV collection medium is added, typically for a 5 day collection. Alternatively, a single medium called HD-EV could be added on day 0 at the same 23,000 cells / ml and microcarrier concentration of 42 mg / mL, with feeds throughout but no change of medium. HD-EV is a chemically defined medium that enables up to 12 days of culture expansion 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). The first experiment to illustrate the challenge, MSCs were expanded in RoosterNourish in 3D bioreactors. During bioreactor culture the EV collection medium was either RoosterCollect-EV (RoosterBio, titled Collection Medium A) or RoosterCollect-EV-Pro (titled Collection Medium B). Cellection Medium B is a richer medium designed to promote cell growth during EV collection leading to greater EV production levels (reference: Trempl, M., Adlerz, K., Rowley, J.A. (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 that in Medium A, cell culture suspension is maintained through day 10. In Medium B, the cell culture is highly viscous by Day 5 and the cells and microcarriers fall out of suspension. Dextran Sulfate, a common cell culture additive to address cell aggregation was added to Medium B to attempt to address the culture failure, but it did not work. The observation was that Medium B increased in viscosity over time, leading to the microcarriers aggregating more, eventually increasing in weight to the point that they cannot be maintained in suspension any longer. When aggregates get too large, internal cells cannot get required nutrients, and EV secreted by internal cells may not make it to the media solution to be harvested. This is demonstrated in Figures 1. Medium viscosity of a typical 10 day bioreactor cell culture illustrating the phenomenon we are addressing was quantified and shown in Figure 2 using an in house viscosity measurement and calculation, reported as Viscosity Index. Briefly; cell culture medium viscosity was measured and recorded as the viscosity index, or [(viscosity timesample) - (viscosity timewater control)] / (viscosity timewatercontrol)]. Viscosity time is the total migration time for 1 mL of liquid solution to proceed through a standard 21-gauge blunt needle from a loaded syringe, attached to a ring stand. Viscosity Index is controlled to account for varying relative humidity and atmospheric conditions. It appears that cells begin secreting high molecular weight Extracellular Matrix (EM) molecules into the culture medium, which can increase liquid viscosity even at low concentrations. This can include collagens, proteoglycans, etc. It has now been demonstrated that novel cell culture additive solutions which include hyaluronidase (HYAL) can decrease and help to maintain low medium viscosities during culture, enabling microcarrier bioreactor cell cultures to be extended without failing, but only when added at the appropriate time and concentrations. To test this, several types of enzymes were added to day 10 viscous media from MSC bioreactor cultures (Figure 3). An initial test was carried out (Figure 3A) which compared the effect on viscosity of adding collagenase, Accumax™, TyrpLE™, or hyaluronidase (isolated from bovine testes, Sigma Alderich) to cells in culture. The results clearly show that hyaluronidase has a significant effect on viscosity by decreasing it dramatically. A significant difference in viscosity, compared to the untreated control, was only seen upon the addition of hyaluronidase (Figure 3A). To further refine these results, varying amounts of hyaluronidase were added (Figure 3B) using both TrypLE and Trypsin as controls. Hyaluonidase at 100 U / mL down to 10 mU / mL decreased the viscosity of the resultant medium, but 0.1 mU / mL was not sufficient to impact viscosity (Fig 3B). TryplLE and Trypsin also did not significantly lower medium viscosity. EV particles were tested from all conditions (Figure 3C) and particle counts (Counted via NTA) were consistent across all conditions illustrating that enzymatic treatment does not destroy the lipid nanoparticles, as expected. Another experiment was performed (Figure 4) comparing a recombinant hyaluronidase (rPH20, ACRO Biosystems) at 3 concentrations (10 mU / mL, 1.0 mU / mL and 0.1 mU / mL) to the bovine-derived hyaluronidase at 1.0 mU / mL). Viscosity Index was calculated in 5 identical bioreactor cultures at days 5, 7, 10 and 12. The Hyaluronidases were added on Day 7. Viscosity in untreated climbed to 1.5 or more on days 10 and 12, while the treated conditions had a dose dependent decrease in viscosity, with the Bovine and recombinant Human hyaluronidase showing equivalent viscosities at the 1.0 mU / mL condition. This is important to demonstrate as the bovine-sourced material would not be appropriate for pharmaceutical manufacturing, and the recombinant human materials could be sourced at GMP quality. Next, the toxicity of hyaluronidase to MSCs in culture was determined. This was a 2D test using media MSFv03, a beta version of the commercially available medium HD-EV (RoosterBio) a chemically defined culture medium designed for cell expansion and EV collection. Hyaluronidase were added to cells at passage 3 on Day 4 of culture, and cell health was monitored (Image at 10X) via morphology for 3 days. Spent medium analysis for p / ml and viscosity was carried out using syringe method. Levels tested were 0, 10, 30, 100, 300, and 500 U / ml hyaluronidase. It was found that hyaluronidase did not have a negative impact on MSC morphology / viability during flask culture, and the cells remained adherent to culture surface and healthy, even up to 500 U / ml. This experiment showed that the addition of HYAL at up to 500 U / ml had no negative effect on the morphology and general health of BM-MSC cells in 2D cell culture. A dose response of hyaluronidase at 10, 100 or 250 U / mL was tested on Day 3 in both flasks and bioreactor culture. Figure 10A shows equivalent cell morphology and growth between all conditions in flasks, Figure 10B shows equivalent cell counts and 10C shows equivalent viability from flask grown cells. 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 at the low concentration of 10 U / mL added on day 3, cells fell off microcarriers and cell growth was impacted as can be seen by the lack of stained cells. Cultures were terminated after day 7. In a final dose finding experiment, MSC microcarrier cultures in HD-EV medium were dosed with 100 U / mL, 1.0 U / mL, 10 mU / mL and 0.1 mU / mL. Viscosity measurements in Fig 8 show that the lowest dose of 0.1 mU / mL did not reduce viscosity, but 10mU / mL and higher did. Fig 12A shows a final dosing study of 1.0, 0.1 and 0.01 mU / mL, with the macroscopic failure of the 0.1mU / mL and 0.01 mU / mL showing in Fig 12B. Thus, 1 mU / mL, or 0.001 U / mL, is thus the recommended working concentration for a standard MSC culture of the conditions stated earlier. Figure 13 A-C demonstrates that 1 mU maintains low culture viscosity, optimal particle production (Fib 13B) and the particle size profiles are all consistent (Fig 13C). It is possible that high concentrations of cells leading to faster hyaluronic acid production can require higher concentrations, but it is important to balance maintaining a low culture viscosity with good cell adhesion to the microcarriers. This data represents a novel use case for hyaluronidase in cell culture. MSC culture viscosity has not been a major issue for MSCs as these cells have not been used as a producer cell to make a biological product such as EVs. Thus, as bioengineers work to maximize cell culture yields and productivity to optimize the cost profile of these expensive products, the need to extend culture life of MSC bioreactors cultures is new. Hyaluronidase addition at these microdosing quantities enables cell growth and EV production for longer than without hyaluronidase treatment, and typical approaches to reducing culture fouling (like dextran sulfate or PEG) did not address the challenge. It was observed that on the harvest day of MSC-EV in a 15L bioreactor (5 day expansion in RoosterNourish / Replenish and medium exchange to RoosterCollect-EV on day 5, 5 day EV collection) that the harvested material was noticeably more viscous than previously generated material from small scale Ambr studies, or even the 250 mL Ambr control in the same experiment. The viscous conditioned medium was treated with 10U / mL hyaluronidase prior to process, and the treatment significantly reduced viscosity of both post-harvest and post-TFF1 material. 10 U / mL of hyaluronidase was tested to determine viscosity and impact on EV counts. To do that, the following experiment was carried out: • Prepare a stock of 1000U / mL hyaluronidase in WFI • Add it to 10mL of samples above and incubate for 30 min at 37°C • Measure particle count and viscosity • For viscosity assay, clamp henke-ject 1mL luerslip syringe to a ring stand and add a 21- gauge blunt tip needle • Add parafilm to the bottom of the syringe. Add 1mL of solution to the syringe. Remove parafilm and let it flow and stop timer when meniscus reaches the last line on the syringe. • Run WFI only as a control • Hyaluronidase stock is stored at -20C • 791 U / mg • Therefore add ~2mg to a tube (actual: 3mg) • Dissolve in WFI – add 2.373mL to dissolve at 1000U / mL • Add 100uL to 10mL for 10U / mL treatment (untreated: add 100uL WFI) • Mix back and forth 15-20 tubes (invert tube) • Add to 37°C incubator for 30min • Take out and measure viscosity The setup can be seen in Table 2. Table 2: Experimental Setup for Harvest / Post-TFF Treatment with Hyaluronidase The results show that viscosity is significantly reduced for samples treated with hyaluronidase. These results can be seen in Figure 8 EV / mL is not changed significantly with treatment. This is important because it demonstrates that a composition comprising hyaluronidase can reduce viscosity in conditioned medium and allow for improved downstream processing. This makes it possible to perform chromatography with higher concentration, less filters required, as well as improved upstream processing (improved suspension, nutrient transfer, recovery). Example 2: Hyaluronidases TABLE 3: Representative Hyaluronidases

[0012] Hyaluronidases exhibit activities that cleave the (154)-linkages between N- acetylglucosamine and glucuronate (EC 3.2.1.35) or comprise hyaluronoglucuronidases (EC 3.2.1.36) with activities which cleave (153)-linkages, or bacterial hyaluronate lyases (EC 4.2.2.1). Exemplar hyaluronidase activities of amino acid and nucleic acid sequences encoded by Homo sapiens hyaluronidase-1 isoform 1 precursor (NP_149349.2), Bos taurus hyaluronidase-1 precursor (NP_001017941.1), and Homo sapiens hyaluronidase PH-20 isoform 2 (NP_001167515.1) are referred to herein. Example 3: Hyaluronidases for Extracellular Vesicle Downstream Processing In standard processes, conditioned medium coming out of bioreactor or culture vessels is first clarified via filtration to remove cellular debris, prior to TFF concentration. The concentrated material is then purified via chromatography, and then once again concentrated via TFF with diafiltration into a final buffer prior to sterile filtration with 0.2 micron filter and vialing. However, during this process, a large amount of EVs are lost. HAase is a novel reagent that streamlines EV downstream processing. Hyaluronidase treatment of conditioned medium (CM), either from flask culture or 3D bioreactor culture significantly decreases pressure build up / filter fouling during the clarification step, enabling increased CM volume to be filtered prior to filter clogging (reaching pressure limit). With the CM traveling through the filter more easily, the EVs do not get caught in the filter which leads to increased total EV yield (Figure 21). This can be seen in Figures 20A-B. . Figure 22 shows that hyaluronidase enables the use of smaller filter cutoff sizes, such as 0.45 um, which improves purification capability. It is noted that filter sizes ranging from 5 micron down to 0.2 micron can be used. Filter size 0.45µm is typical during bioprocessing before TFF and chromatography to streamline these unit operations, as well as before 0.2µm (sterile) filtration. This is required for optimal performance of downstream processing steps (TFF, Chromatography, Formulation / Fill). Note, the low amounts of hyaluronidase added during bioreactor culture, added at levels (~ 1mU / mL of culture volume) so as not to impact cell adhesion and growth, is not sufficient to facilitate the downstream purification, and approximately 100 fold high concentrations (100 mU / mL) are optimal for downstream purification. Hyaluronidase can be added to the bioreactor prior to CM collection, or immediately before clarification occurs. Figure 23 shows that treatment of conditioned medium with hyaluronidase maintains EV quality attributes. For example, tetraspanin marker expression is maintained. Furthermore, data have shown that hyaluronidase treatment does not detach cells from microcarriers. Figures 24, 25, and 26 show results at various doses, incubation times, and temperatures. It has been found that 0.1 U / mL gives a balance of low pressure buildup of filter and high EV yield comparable to 5-10 times more hyaluronidase. As GMP grade hyaluronidase is expensive, it is important to use the least amount to achieve good processing and yield. In summary, the addition of hyaluronidase to filtration during downstream processing (DSP) of EVs provides the following advantages: • Enhances EV yield through filtration • Increases process efficiency (greater #EVs recovered) • Enables use of smaller cutoff filters (tested down to 0.45µm) • Provides ability to clarify impurities >0.45µm (a requirement for DSP) without significant EV loss. The ability to use smaller cutoff filters enables more efficient TFF, as less debris to clog TFF filters. • Enables greater filter loading capacity. Great filter loading means more volume of CM can go through a filter of set size. Filters increase in cost as surface area increases, so smaller filters are more cost effective. The above advantages culminate in a cumulative ~10X greater particle yield (after a complete purification process as seen in Fig 18A) with significant cost reductions in GMP manufacturing operations. Greater yields and lower costs lead to significant cost benefits in therapeutic development. For pressure vs. loading results in 3D, the untreated sample reached maximum pressure at ~32 L / m2at which point the filter needs to be changed out (if it fully fouled, or clogged). If a filter fouls and reaches max pressure during processing then a second filter needs to be attached, doubling the disposable cost at that unit operation. In this example, in ~10 min of process time only ~15mL of sample could be filtered. Pressure vs loading curves show a clear impact of treatment in improving filtration with more CM being processed without filter fouling. Furthermore, treatment with any tested Agent V (hyaluronidase) concentration increases yield by ~2.8X. This effect is saturated at the 0.1 U / mL condition. These results can be seen in Figures 30 and 31. Example 4: Human Dermal Fibroblast Expansion hMSCs clump and increase medium viscosity in longer term 3D cultures with a goal of maximizing EV collection leading to culture failure (microcarrier aggregates falling out of solution). Viscosity increase and aggregation can be mitigated by hyaluronidase addition (Agent V) during culture. In this example, it is demonstrated that this viscosity build up is not only a challenge with MSCs, but other primary cells such as fibroblasts. It was determined that viscosity builds in 3D bioreactor cultures of human dermal fibroblasts (HDFs), and that hyaluronidase is able to reduce viscosity and significant microcarrier clumping. The experimental setup is shown in Figure 31. HDFs were expanded and seeded into spinner flasks with Corning Synthemax II microcarriers (23K cells / mL, 42 mg microcarriers / ml in a total volume of 100 mL) in RoosterNourish. Medium was exchanged to EV-Pro collection medium on day 5 for 5 days of EV production. Cell counts, medium viscosity and EV counts were monitored over time. Figure 32 shows that fibroblasts expanded well and microcarrier aggregates were quite large, larger than MSC aggregates. Medium viscosity increased substantially, but cell remained viable and secreted EVs. Figure 33 shows high cell counts and high viscosity with EV production. The viscosity was greater than that seen in MSC cell culture. Day 10 viscous medium was treated with Agent V (hyaluronidase) at multiple concentrations, and TrypLE was used as a control. Medium viscosity was measured, as were EV counts. Agent V (hyaluronidase) was used at a stock concentration of 100 U / mL. TrypLE was added as 10% volume 10x TrypLE as a test and control. Highly viscous medium had viscosity reduced by HAase in a dose dependent fashion, similar to MSC medium, and the protease TrypLE did not impact viscosity. EV Particle count and size profile was not impacted by Agent V. (Figures 34 and 35). This data supports that the phenomenon of media viscosity increase is not only an MSC phenomenon, and Agent V can both reduce viscosity of CM, benefiting long term microcarrier culture of multiple cell types, and that post harvest treatment with Agent V can increase EV yield after filtration. In conclusion, HDFs proliferated well in RoosterNourish growth medium in 3D microcarrier spinner flask culture. Large microcarrier aggregates formed, which were larger than in MSC 3D cultures. HDF culture medium viscosity built during the EV collection phase, illustrating that this phenomenon is not specific to only MSCs. Hyaluronidase reduced viscosity in a dose-dependent manner while the protease, 1x TrypLE did not. This data supports that the phenomenon of media viscosity increase is not only an MSC phenomenon, and Agent V can support long term microcarrier culture of multiple cell types. Example 5: EV Yields from Conditioned Medium collected from HEK293 Cells hMSC conditioned medium (CM) has EVs, that when filtered to clarify or sterilize, cause many of the EVs to be lost. It appears that the mechanism is that soluble hyaluronan cannot get through filters even when viscosity is not an issue, trapping EVs while fouling the filter. Treatment of culture media with HAase can break down the hyaluronan into smaller fragments enabling them and more EVs to make it through the filter. In this example, it is shown that HAase treatment of culture media from cultures of ExpiHEK293F cells enables higher EV yields when sent through 0.45 micron filter. Expi293F cells are a high protein expressing HEK 293 cell line marketed by Gibco. Culture media was collected from cultured Expi293 cells. This experiment determines the effectiveness of Agent V to reduce any viscosity, and / or increase EV yield after filtration. The experimental design is shown in Figure 36. Figure 37 shows the results. Interestingly, it was found that HEK medium was not viscous, but EV recovery was increased from 73% to 100% compared to a control.

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Claims

WHAT IS CLAIMED IS:

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

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

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

4. The method of claim 1, wherein the HAase is introduced to the liquid medium concurrently with introduction of cells.

5. The method of any one of claims 1-4, wherein, upon addition of HAase, viscosity of the liquid medium is decreased by 10% or more.

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

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

8. The method of claim 7, wherein in an adherent culture, the cells may be adherent to plastic, to glass, to chemically derivatized surfaces, to polypeptides, or to polysaccharides.

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

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

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

12. The method of any one of claims 1-10, wherein the cells are HEK-293 or HEK-293T cells or derivatives or sublineages thereof.

13. The method of any one of claims 1-10, wherein the cells are mesenchymal stem cells, multipotent stromal cells, mesenchymal stromal cells, or mesenchymal progenitor cells or derivatives or sublineages thereof.

14. The method of any one of claims 10-13, wherein the cells are isolated from umbilical cord, bone marrow, adipose tissue, adipose stem cells (ASCs), adipose tissue, adipose stromal vascular fraction, soft tissue organs, cartilage, perivascular tissues, blood vessels, heart, placenta, ectopic pregnancy tissue, peripheral blood, cord blood, teeth, or menstrual blood.

15. The method of any one of claims 1-14, 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 of any one of claims 1-16, wherein HAase is introduced to the liquid medium at a temperature of about 37°C or 25°C (room temperature), and / or across a range spanning between 1°C to 50°C.

18. The method of any one of claims 1-17, wherein HAase is applied to the liquid cell culture media to improve co-optimized conditions including varied agitation rates and media compositions, wherein said addition can reduce viscosity and / or fouling of the cell culture quality and loss of cell suspensions.

19. The method of any one of claims 1-18, wherein the cells in the cell culture are used for the bioproduction of molecules or particles for therapeutic, veterinary, diagnostic, human wellness, cosmetic, and / or theragnostic use.

20. The method of any one of claims 1-19, wherein the HAase is derived from a cell or synthetically manufactured.

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

22. The method of claim 21, wherein the cell which produces HAase is one of the cultured cells in the media.

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

24. The method of claim 23, wherein the mammalian cell originated from mammalian tissue or a mammalian organism.

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

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

27. The method of claim 26, wherein the native HAase comprises at least 60% identity to any of SEQ ID NOS: 1-3.

28. The method of claim 26, wherein the HAase is identical to the amino acid sequence associated with any of SEQ ID NOS: 1-3.

29. The method of any one of claims 21-28, wherein the engineered cell comprises an expression system that is synthesized from and / or comprised of recombinant nucleic acids.

30. The method of claim 29, wherein the HAase is derived from coding DNA sequences (cds) encoding wild type, point mutated, deletion mutated, insertionally mutated, or codon- optimized expression systems to produce the HAase.

31. A method of improving properties of liquid cell culture media, the method comprising culturing cells in the liquid medium, removing said cells, and then introducing HAase to the liquid medium after cells are removed.

32. The method of claim 31, wherein HAase reduces viscosity of the liquid 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, including extracellular vesicles (EVs), exosomes, microvesicles, cellular ghosts, secretory granules, or virus particles.

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

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

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

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

38. The method of claim 37, wherein the liquid medium is concentrated 2-100 fold via diafiltration, centrifugation, or ultracentrifugation.

39. The method of any one of claims 31-38, wherein the HAase is derived from a cell or synthetically manufactured.

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

41. The method of claim 40, wherein the cell which produces HAase is one of the cultured cells in the media.

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

43. The method of claim 42, wherein the mammalian cell originated from mammalian tissue or a mammalian organism.

44. The method of claim 42 or 43, wherein the mammal is Homo sapiens or Bos taurus.

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

46. The method of claim 45, wherein the HAase is 60% or more identical to the amino acid sequence comprising any of SEQ ID NOS: 1-3.

47. The method of any one of claims 40, wherein the engineered cell comprises an expression system that is synthesized from and / or comprised of recombinant nucleic acids.

48. The method of claim 39, wherein the HAase is derived from coding DNA sequences (cds) encoding wild type, point mutated, deletion mutated, insertionally mutated, or codon- optimized expression systems to produce the HAase.

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

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

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

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

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

54. The method of any one of claims 31-53, wherein HAase is added before clarification takes place.

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

56. The method of claim 55, wherein filters of pore sizes between 0.45um and 100 µm are used in post-cell culture downstream processing.

57. A composition comprising liquid cell culture media and exogenously added hyaluronidase.

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

59. The composition of claim 58, wherein the cells are mesenchymal stem cells (MSCs).

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

61. The composition of any one of claims 57-60, wherein the cells are attached to a microcarrier in a bioreactor, the cells are at a density of 50,000 to 5,000,000 cells / mL, where EVs are within a concentration of 1E8 to 1E11 particles / mL, and exogenous hyaluronidase is present at a concentration between 0.0001 U / mL and 100 U / mL 62. The composition of claim 60 or 61, wherein cells have been removed from the liquid cell culture media.

63. The composition of claim 62, wherein the liquid cell culture media comprises one or more products secreted by the cells, including extracellular vesicles (EVs), exosomes, microvesicles, cellular ghosts, secretory granules, or virus particles.

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

65. The kit of claim 64, wherein the HAase is within the liquid cell culture media.

66. The kit of claim 64 or 65, wherein the liquid cell culture media can be used for cell expansion or collection of conditioned media.

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

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

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