Methods of culturing pluripotent stem cells by perfusion

Abstract

The present invention relates to a method of culturing pluripotent stem cells by perfusion, the method comprising culturing the pluripotent cells in a cell culture medium and applying shear to the medium. Cells obtained by the methods, as well as products, such as food products, comprising the cells obtained by the method are also described herein.

Description

[0001] Methods of culturing pluripotent stem cells by perfusion

[0002] Field of the invention

[0003] The present invention relates to a method of culturing pluripotent stem cells by perfusion.

[0004] Background of the invention

[0005] According to the most recent United Nations estimations, the current world population is 7.9 billion in July 2022 [https: / / www.worldometers.info / es / poblacion-mundial / #ref-1] and it is expected to reach 10 billion around the year 2056. This increase will be heterogeneously distributed around the globe, with nine countries covering half the projected growth of the global population in the next 30 years, including India, Nigeria, Pakistan, Egypt, and the United States of America. Population and economic growth are major drivers of increased meat consumption. According to the United Nations Food and Agricultural Organization (FAO, https: / / www.oecd- ilibrary.org / agriculture-and-food / oecd-fao-agricultural-outlook-2022-2031_f1 b0b29c-en), an estimated growth of 15% in global meat consumption is projected by 2031 . On the other hand, the correlation between income growth and higher meat consumption is clearly demonstrable at lower income rates but once consumers reach an adequate standard of living, they become more sensitive to environmental, ethical, and animal welfare and health concerns.

[0006] For this reason, there is a growing interest in finding alternative protein sources which ideally will be sustainable and will contain the nutrients normally provided by meat in the human diet. Cultured (or cultivated) meat arises as another alternative to traditional animal agriculture that aims to produce the skeletal muscle and adipose tissues that normally comprise animal meats, except using in vitro tissue and biological engineering techniques. Despite efforts to develop robust protocols for scalable generation of animal cell types from easily accessible and renewable sources, protocols for scaling up production often remains cumbersome, lengthy, and difficult to reproduce and / or has not been established yet. Thus there remains a need for culture methods that allow scalability of stem cells.

[0007] Summary of the invention

[0008] In a first aspect, the invention relates to a method for reducing the size of cell aggregates, for example pluripotent stem cell aggregates, during cell culture the method comprising culturing the pluripotent cells by perfusion, such as for example continuous perfusion, in a cell culture medium and applying shear to the medium.

[0009] In one embodiment, the shear rate is applied in the range from about 90 sec1to 2829 sec1, preferably about 1100 sec-1 to 2200 sec-1 , or more preferably about 1500 sec1to 2000 sec1. In one embodiment, during culture shear stress is applied in the range from about T10-2Pascal to about 1 -10 ° Pascal.

[0010] In one embodiment, the cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, somatic cell lines and immortalized cell lines. In one embodiment, the cells produced by the method are suitable for human and nonhuman dietary consumption. In one embodiment, the method is forthe proliferation ofthe pluripotent cell. In one embodiment, the method as described herein further 10, further comprises the step of: - incorporating the cultured cell into a food product for animal, preferably human, consumption.

[0011] In a second aspect, the invention provides for a cell obtainable by or obtained by the method as described herein.

[0012] In a third aspect, the invention provides for a cultured meat product for animal, preferably human, consumption, comprising at least one cell obtained by the method as described herein, optionally further comprising (cultured) mammalian myocytes.

[0013] In a fourth aspect, the invention provides for a cultured fat product or cultured muscle product for animal, preferably human, consumption, comprising at least one cell obtainable by a method as described herein.

[0014] In a fifth aspect of, the invention provides for the use of a cell obtained according to the method as described herein or use of the method as described for tissue engineering, optionally for the production of cultured meat.

[0015] Description of the invention

[0016] Definitions

[0017] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the method.

[0018] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0019] As used herein, the term "and / or" indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0020] As used herein, with "At least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ... ,etc.

[0021] The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1 % of the value.

[0022] “Expansion” refers to the proliferation of a cell with or without differentiation and may include no passaging, one passage or more than one passage and / or serial passages. As used herein, a “medium” or “cell culture medium” refers to an aqueous based solution that provides for the growth, viability, or storage of cells. A medium as contemplated herein can be supplemented with one or more nutrients to promote the desired cellular activity, such as cell viability, growth, proliferation, differentiation of the cells cultured in the medium. A medium, as used herein, includes a serum replacement, a medium supplement, a complete medium or a cryopreservation medium. The pH of a culture medium should be suitable to the microorganisms that will be grown. Most bacteria grow in pH 6.5-7.0 while most animal cells thrive in pH 7.2-7 .4.

[0023] “Aggregate” refers to an association of cells in which the association is caused by cell-cell interaction rather than adherence to a substrate. In an aggregate, two or more cells associate with each other by biologic attachments to one another. This can be through surface proteins, such as extracellular matrix proteins. In one embodiment, cells spontaneously associate in suspension to form cell-cell attachments independent of any adherence to a surface. In one embodiment, cells can be initially grown on a substrate where some cells associate with (adhere to) the substrate but further growth forms cell-cell associations (aggregation) that do not depend on association (adherence) of the further-grown cells with the substrate. A cellular feeder layer is also considered a substrate. So attachment of cells to a feeder layer is also a form of adherent culture (not an aggregate) since attachment of the cells is not to each other but to the cells in the feeder layer.

[0024] The terms “cultured” and “cultivated”, in the context of “cultured meat” and "cultivated meat”, “cultured fat / muscle”, cultivated fat / muscle” may be used interchangeably herein.

[0025] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0026] During the culture of cells, such as pluripotent cells, it was observed that the cells started to form larger aggregates over time. Due to this aggregation, when cells proliferate, this results in the increase of the diameter of the aggregate. In order to keep cells proliferating at desirable rates, nutrient limitations need to be prevented. Unfortunately, since aggregates have no vascular system, they will show nutrient limitations in the core of the aggregate due to diffusion limitations. Thus, these larger aggregates are a problem as they hamper cell growth and ultimately cause cell death (see Fig. 1). The formation of large aggregates is one of the reasons that scaling-up pluripotent cell culture is hampered. The inventors of present application have surprisingly found that applying shear stress during the culture of pluripotent cells reduces the size of the aggregates which allows for better growth and expansion of the cells.

[0027] Accordingly, the invention provides for a method for reducing the size of cell aggregates, for example pluripotent stem cell aggregates, during cell culture, the method comprising culturing the cells by perfusion in a cell culture medium and applying shear to the medium. The perfusion process may be a continuous perfusion process.

[0028] As used herein “perfusion” refers to the process of keeping culture cells alive by continuously feeding the cells with fresh media and removing spent media while keeping cells in culture. Perfusion is typically a continuous process. Perfusion culturing includes, but is not limited to continuous flow and semi-continuous flow, for example step-wise flow or staggered flow. In one embodiment of the method as described herein, during the expansions and / or proliferation of the cells, the average diameter of each expanded cell aggregate is no more than about 250 micron in size. In one embodiment of the method as described herein, during the expansions and / or proliferation of the cells, the average diameter of each expanded cell aggregate is no more than about 200 micron in size. In one embodiment of the method as described herein, during the expansions and / or proliferation of the cells, the average diameter of each expanded cell aggregate is no more than about 150 micron in size. In one embodiment of the method as described herein, during the expansions and / or proliferation of the cells, the average diameter of each expanded cell aggregate is no more than about 100 micron in size. In one embodiment of the method as described herein, during the expansions and / or proliferation of the cells, the average diameter of each expanded cell aggregate is no more than about 50 micron in size. It will be appreciated that where size is indicated throughout the document, the size refers to an average size in a population of aggregates.

[0029] In mechanics, shear forces are a common phenomenon. A shear force is understood to exist when there is a first force acting on (part of) a composition in a first direction, and a second force acting on (part of) the composition that is stationary or moving in a second non-aligned direction.

[0030] Shear rate is the rate at which a progressive shearing deformation is applied. For a simple case, this can be the gradient of velocity in a flowing material. Shear rate is expressed in “reciprocal seconds, i.e. in “sec-1".

[0031] In one embodiment, the a shear rate applied to the cultured pluripotent cells in the range of about 990 sec-1 to 2829 sec-1 , about 1100 sec-1 to 2200 sec-1 , or about 1500 sec-1 to 2000 sec- 1 . In preferred embodiments, the shear rate is about 1800 sec-1 , such as about 1839 sec-1 .

[0032] Shear stress (often denoted by the Greek symbol “tau” or “r” ) is the component of stress coplanar with a material cross section. It arises from the shear force and more specifically from the component of force vector parallel to the material cross section. Average shear stress refers to the force applied per unit area and can be calculated with formula (I) below: T = F / A (I) wherein:

[0033] T = the average shear stress;

[0034] F = the force applied;

[0035] A = the cross-sectional area of material with area parallel to the applied force vector. Preferably a shear stress is applied in the range from about 1x10-1, about 10-1 5- about 1 -10’2Pascal to about 1 10° Pascal.

[0036] In one embodiment of the invention, shear stress is applied by use of an Alternating Tangential Flow (ATF). The ATF system uses hollow fibres over which the shear is homogeneously distributed. Due to slow alternating flows (every ~10 seconds), the change in pressure is relatively low which also results in lower shear stress peaks and an overall lower stress rate (990 - 2829 sec- 1). Preferably, a cell used in the invention may be a “pluripotent stem cell”. As used herein, the term "pluripotent stem cells" includes embryonic stem cells, embryo-derived stem cells, epiblast- derived stem cells, induced pluripotent stem cells and somatic cells, regardless of the method by which the pluripotent stem cells are derived. Accordingly, in certain embodiments the pluripotent stem cell is selected from the group consisting of embryonic stem cells, embryo-derived stem cell lines, epiblast-derived stem cell lines, induced pluripotent stem cells, embryonic cell lines, somatic cell lines and and immortalized cell lines. In certain embodiments, the pluripotent stem cells are epiblast-derived stem cells (EpiSC). In certain embodiments, pluripotent stem cells express one or more markers selected from the group consisting of: OCT-4, Sox2, Klf4, c-MYC, Nanog, Lin28, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Exemplary pluripotent stem cells can be generated using, methods known in the art. "Induced pluripotent stem cells" (iPS cells or iPSC) can be produced by protein transduction of reprogramming factors in a somatic cell.

[0037] The pluripotent stem cell according to the invention can be from any species. Embryonic stem cells have been successfully derived in, for example, mice, multiple species of non-human primates, and humans, and embryonic stem-like cells have been generated from numerous additional species. Thus, one of skill in the art can generate pluripotent stem cells, such as embryonic stem cells, embryo-derived stem cells, epiblast-derived stem cells (EpiSC), from any species, including but not limited to human, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrel, guinea pig, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like.

[0038] Similarly, iPS cells can be from any species. These iPS cells have been successfully generated using mouse and human cells. Furthermore, iPS cells have been successfully generated using embryonic, fetal, newborn, and adult tissue. Accordingly, one can readily generate iPS cells using a donor cell from any species. Thus, one can generate iPS cells from any species, including but not limited to, human, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like.

[0039] In certain embodiments, the pluripotent stem cell according to the invention, or for use in the invention is an animal cell. In certain embodiments the pluripotent stem cell according to the invention, or for use in the invention if from an edible animal species.

[0040] In certain embodiments, the cell is from a mammal, preferably a non-human mammal. Preferably, the cell is an animal cell. In certain embodiments cell according to the invention is from an edible non-human animal species.

[0041] Accordingly, in one embodiment, the cells produced by the method as described herein are suitable for human and non-human dietary consumption.

[0042] In certain embodiments, the pluripotent stem cells are of a livestock or poultry species. Poultry species include but are not limited to domestic chicken, turkeys, ducks, geese and pigeons. In certain embodiments, the cells originate from common game species such as wild deer, gallinaceous fowl, waterfowl and hare.

[0043] Livestock species include but are not limited to domestic cattle, pigs, sheep, goats, lamb, camels, water buffalo and rabbits.

[0044] In certain embodiments, the cell is a cell that is naturally capable of lipid accumulation such as a fat cells (adipocytes) or precursor cells thereof (such as fibro-adipogenic progenitor cells, lipoblasts, adipoblasts and, pre-adipocytes) or liver cells (hepatocyte) or precursor cells thereof (such as pre-adipocytes, hepatoblasts and pre-heptatocytes).

[0045] In certain embodiments, the cells are derived from an immortalized cell line. Exemplary cell lines include, but are not limited to, 3T3-L1 (mouse pre-adipocytes), buffalo rat liver cells (BRL 3A), chicken liver cells (LMH),

[0046] In certain embodiments, the cell is an adipocyte originating from a domestic pig or domestic cattle.

[0047] In certain embodiments, the cell is an hepatocyte originating from a duck or a goose.

[0048] In certain embodiments, the cell is a cell that is a muscle cell (myocyte), such as a skeletal muscle cell, or a or precursor cell thereof.

[0049] In certain embodiments, the cell is a muscle cell originating from a domestic pig or domestic cattle.

[0050] In certain embodiments, the cell is a pluripotent cell. Preferably the pluripotent cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines. Preferably, when a pluripotent cell is cultured, the pluripotent stem cell is a non-human animal cell, such as a porcine or a bovine pluripotent stem cell. Most preferably, a porcine pluripotent stem cell. Such a cell may be a fat cell or a precursor cell thereof. Such a cell may be a muscle cell or a precursor cell thereof.

[0051] In certain embodiments, the stem cell according to the invention is a porcine epiblast stem cell (pEpiSCs) or a bovine epiblast stem cell (pEpiSCs). Such a cell may be a fat cell or a precursor cell thereof. Such a cell may be a muscle cell or a precursor cell thereof.

[0052] Preferably a pluripotent stem cell according to the invention, or for use in the invention, is not a human cell.

[0053] In certain embodiments, the cells as used in the methods as described herein are described in patent application nos. W02024 / 084082, WO2024 / 170696, WO2024 / 170702 and W02024 / 252001 . Any cell described in any one of those patent applications may be useful in the methods of the invention for culturing cells.

[0054] In one embodiment, the method according to the invention is for the proliferation (i.e. expansion) of the pluripotent cell.

[0055] In one embodiment, the method as described herein can be used for differentiation the pluripotent cell.

[0056] In certain embodiments, the culture medium is a basal medium that is supplemented with said medium components. The term ‘basal medium’, as used herein, includes reference to a liquid medium that supports cellular growth by providing essential components for growth. A basal medium may be provided in liquid or powdered format. A basal medium that is not supplemented with any compound may enable cellular growth, but supplementation may be required for growth depending on the cell type. A basal medium may be supplemented with one or more components selected from the nonlimiting group consisting of amino acids, lipids, sugars, carbohydrates, anions, cations, buffering agents, colorants, vitamins, antioxidants, hormones, enzymes, proteins and trace elements. In some embodiments, the basal medium as disclosed herein is a commercially available basal medium such as DMEM (Dulbecco’s Modified Eagle Medium) and Ham’s F- 12. In some embodiments, the basal medium is the medium described in any of patent application nos. W02024 / 084082, WO2024 / 170696 and WO2024 / 170702 and W02024 / 252001 , which are incorporated by reference herein. Any of the medial described in any one of those patent applications may be used as basal medium in the methods of the invention.

[0057] In certain embodiments, the methods as described in the various embodiments of the invention further comprises the step of:

[0058] - incorporating the cultured cell into a food product for animal, preferably human, consumption.

[0059] In a second aspect, the invention provides for a cell obtainable by or obtained by the methods as described in the various embodiments as described herein. In one embodiment, cells obtained by the method as described herein are suitable for human and non-human dietary consumption.

[0060] In yet a further aspect, the invention provides for a food product (also referred to as “foodstuff’) comprising the cells produced, obtained by the methods and / or cultured in any of the mediums as described herein. In certain embodiments, the food product is for animal, preferably human, consumption.

[0061] A food product of the present invention may comprise one or more of minerals, synthetic substances, flavors, texture enhancers, nutritional additives, preservatives, and fats. In an aspect of the invention, the flavors are selected from one or more of essential oils, oleoresin (ESO), enzymes (ENZ), natural substances and extractives (NAT), non-nutritive sweetener (NNS), nutritive sweetener (NUTRS), herbs, spices, natural seasonings & flavorings (SP), and synthetic flavors (SY / FL), fumigant (FUM), artificial sweeteners and yeast extract. In another aspect of the present invention, the texture enhancers are selected from one or more of pureed plant material, guar gum, cellulose, hemicellulose, lignin, beta glucans, soy, wheat, maize and rice isolates and beet fiber, pea fiber, bamboo fiber, plant derived fiber, plant derived gluten, carrageenan, xanthan gum, lecithin, pectin, agar, alginate, natural polysaccharides, grain husk, calcium citrate, calcium phosphates, calcium sulfate, magnesium sulfate and salts. In another aspect of the present invention, the nutritional additives are selected from one or more of trace elements, bioactive compounds, endogenous antioxidants, A, B-complex, C, D, E vitamins, zinc, thiamin, riboflavin, selenium, iron, niacin, potassium, phosphorus, omega-3, omega-6, fatty acids, magnesium, protein, amino acids salt, creatine, taurine, carnitine, carnosine, ubiquinone, glutathione, choline, glutathione, lipoic acid, spermine, anserine, linoleic acid, pantothenic acid, cholesterol, Retinol, folic acid, dietary fiber and amino acids. In yet another aspect of the present invention, the fats are selected from one or more of saturated, monounsaturated, polyunsaturated fats, corn oil, canola oil, sunflower oil, safflower oil, olive oil, peanut oil, soybean, flax seed oil, sesame oil, canola oil, avocado oil, seed oils, nut oils, safflower and sunflower oils, palm oil, coconut oil, omega-3, fish oil, lard, butter, processed animal fat, adipose tissue, cellular agriculture derived fat essential oil and oleoresin. In another aspect of the present invention, the preservative and / or antioxidant is selected from one or more of: sodium salt, chloride salt, iodine salt. Nitrates, nitrosamines. butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), sodium benzoate, potassium benzoate and benzene ascorbic acid, citric acid, potassium, monosodium glutamate (MSG), sulphur dioxide, sulphites, antibiotics. It is noted here that any one additive, flavor, texture enhancer, nutrient additive, fat / oil and / or preservative / antioxidant may supply more than one attribute to food product of the present invention.

[0062] In certain embodiments, for example where the cell that is cultured is a fat cell or a precursor cell thereof, the food product of the present invention may further comprise (cultured) myocytes. In certain embodiments, the food products comprises the cultured myocytes as described in patent application nos. WO2024 / 170696 and WO2024 / 170702. Any cell described in any one of those patent applications may be useful in a method of the invention for culturing cells and in a food product of the invention.

[0063] In certain embodiments, for example where the cell that is cultured is a muscle cell or a precursor cell thereof, the food product of the present invention may further comprise (cultured) adipocytes. In certain embodiments, the food products comprises the cultured adipocytes as described in patent application no. W02024 / 084082. Any cell described in any one of those patent applications may be useful in a method of the invention for culturing cells.

[0064] That is to say, a food product of the invention may comprise one or more types of cell, for example two or more types of cell. At least one of the cell types is a cultured cell. A food product of the invention may comprise two or more types of cultured cell, one or two or more of which may be obtained using a method of the invention.

[0065] In certain embodiments, the food product is a cultured meat product, for example a cultured fat product or a culture muscle product.

[0066] The cultured meat product may be a structured product. Optionally, the product is structured with scaffolding. In one embodiment the structured product is free of scaffolding and the structure is provided by the cells as obtained by the methods as described herein.

[0067] In yet a further aspect, the invention provides for a cultured fat product or a cultured muscle product for animal, preferably human, consumption, comprising at least one cell obtainable by a method as described herein.

[0068] In yet a further aspect, the invention provides for a use of the cell obtained or obtainable by the method as described herein for tissue engineering, optionally for the production of cultured meat. Description of the figures

[0069] Figure 1 : Cell-aggregate staining. Nuclei were visualized with DAPI (blue), dead cells (green) are visualized with LIVE / DEAD™ Fixable Green stain. Image was taken with a EVOS M7000 microscope.

[0070] Figure 2: Repeated fed-batch culture for 5 days. A) shows the median cell aggregate diameter over time in pm. B) Cell growth rate over time (p, day-1) C) Proliferation markers (OCT4 and SSEA4) over time.

[0071] Figure 3: Cell culture under perfusion with stainless steel filter. A) shows the median cell aggregate diameter over time in pm. B) Cell density over time.

[0072] Figure 4: Cell culture with ATF. A) shows the median cell aggregate diameter over time in pm. B) Cell growth rate over time (p, day-1) C) Total cells over time.

[0073] Figure 5: Cell culture with peristatic pump recirculation cell viability after 30min of culture.

[0074] Examples

[0075] The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

[0076] Stem cells in suspension will grow in aggregates / spheroids and not as single cells. As cells continue to proliferate, the diameter of the aggregate continues to increase. Cell aggregates have no vascular system, so nutrients present in the culture medium cannot reach the core of cells causing the cells at the center of the aggregate to die, which ultimately results in the death of the entire aggregate. Therefore, to have optimal cell proliferation, it is of importance to reduce the size of aggregates in cell culture. From prior internal research it was observed that when an aggregate reached a diameter of ~300 pm, the cells at the center of the aggregate start to die (See Fig 1). Thus, to enable the run of a process, ideally continuous, in which cells can grow in exponential phase for multiple weeks without a dead core to appear, aggregate size control must be in place.

[0077] Here we describe a way of controlling the aggregate size while maintaining acceptable growth rates of the stem cells. It was observed that a shear stress of about (990 - 2829 sec1) was capable of keeping aggregates at a stable size thereby improving cell culture conditions.

[0078] Materials and Methods

[0079] The cells used for these experiments have been described in patent applications EP23156930.2 and EP23156923.7 both of which are incorporated herein by reference herein.

[0080] The following bioreactors have been used to culture the cells using the MyControl control system from Getinge Applikon:

[0081] • Single use Appliflex (500 ml and 3L) from Getinge Applikon

[0082] • Multi-use glass 3L Getinge Applikon bioreactor All bioreactors control pH, dissolved oxygen (DO), temperature and impeller speed and are run under beforementioned control levels.

[0083] The following culture methods have been used:

[0084] Fed-batch

[0085] Cells are inoculated as single cells in bioreactor with inoculation media. Over time, the bioreactor is filled with feed media and cells will form aggregates. Once the maximal bioreactor volume is reached, the cells are harvested and require a single celling procedure to be seeded into a new bioreactor.

[0086] Repeated fed-batch

[0087] Similar to the fed-batch as described herein but once the maximal bioreactor volume is reached, a part of the broth is removed which allows the fed-batch to continue again for a certain period of time.

[0088] Perfusion

[0089] Cells are inoculated as single cells or small aggregates (depending on the specific experiment) with inoculation media. Over time, the bioreactor is filled with feed media and cells will form aggregates. Once the desired working volume is reached, perfusion will be started. Here the flow rate of broth out of the bioreactor will be equal to that of the feed media flowing in. In order to remove very little cells, different cell retention devices can be used. This allows cell to be retained in the bioreactor while spent media can be removed. In these examples two different setups are compared: ATF1 filter (Repligen, USA) and a submerged stainless steel stationary dead-end filter are used.

[0090] Example 1 : Repeated fed-batch - no shear applied

[0091] A repeated fed-batch experiment was set-up in a 500 ml single use Appliflex bioreactor. Cells were grown in a regular fed-batch phase for 5 days. After that, the bioreactor was partially harvested (50% of volume removed) and filled again over the next 24 hours to repeated the process for 4 days (process time in total is 9 days). During these 9 days, samples were taken to analyze aggregate size distribution, growth rate and to quantify the change in several pluripotency markers over time. Results

[0092] Once the aggregates grow larger than 250 pm (from day 6) (Fig. 2A), a large reduction in growth rate is observed (Fig. 2B) Moreover, from day 8 the impact of the large aggregate size on the cells’ health is also clearly visible from the proliferation markers, both OCT4 and SSEA4 show a significant reduction in presence (Fig. 2C). This shows that cell culture for longer than a week is not feasible which is a significant problem which limits scaling up the cell production.

[0093] Example 2: Perfusion with stainless steel filter - no added shear applied

[0094] Next a perfusion set-up was tested to measure if the above issues of cell aggregation and resulting limiting culture times could be resolved. In this setup a glass 3L vessel from Applikon Getinge was used and the bioreactor was run for 7 days: a 2-day fed-batch period in which the bioreactor was filled with feed media to the desired volume. After these two initial days, perfusion was started and the processed was followed for another five days. Here the media was removed through the stainless steel filter with a pore size of 10 |j.m that retains the cells and allows spent media to pass through.

[0095] Results

[0096] As can be seen, cell aggregates steadily increases over time (Fig. 3A). As soon as the median cell aggregate size exceeds 250|j.m (see day 6 Fig. 3A), a large drop in cell density is observed which is indicative of a reduction in growth and / or cell death (Fig. 3B).

[0097] Example 3: Perfusion with ATF

[0098] ATF uses 60 cm hollow fibres over which the shear is homogeneously distributed. Due to slow alternating flows (every ~10 seconds), the change in pressure is relatively low which also results in lower shear stress peaks and an overall lower stress rate (990 - 2829 sec1). The process was very comparable to the one described in Experiment 2. However, perfusion was started on day 5 instead of day 2 and the run was continued for a total of 12 days.

[0099] Results

[0100] ATF was turned on at day 5, as can be seen, this has an immediate effect on the median aggregate size as this is reduced to 50-75 pm (Fig. 4A) . This median aggregate size is maintained for at least 7 days and the growth rate is continued on the same pace (Fig. 4B-4C). This strongly indicates that the shear stress applied by the ATF is controls the aggregate size in such a way that the cells are not experiencing too much shear that would impact their growth performance.

[0101] Example 4: Peristaltic pump recirculation

[0102] Next, cells were cultured under fed-batch conditions in a 500 ml Appliflex bioreactor and then recirculated in the presence of a peristaltic pump (a D-SP086 with Masterflex 06402-25 Tygon tubing at ~150 ml / min). In a peristaltic pump the pressure differences are significantly higher and more frequent (multiple times per sec) and therefore the cells also tend to experience very high peaks of shear stress and an overall higher shear rate of about 3000 sec1.

[0103] Results

[0104] Over time the viability of the cells in the bioreactor were measured and showed a reduction from 84% to 64% within 30 minutes of recirculation (Fig. 5). This suggests that this amount of shear is too high and or / too intense and has a negative effect on the health of the cells and could therefore never be used for successful aggregate size reduction / control.

[0105] Embodiments

[0106] The present invention provides at least the following numbered statements / embodiments:

[0107] 1 . A method for reducing the size of stem cell aggregates during cell culture the method comprising culturing the pluripotent cells by perfusion in a cell culture medium and applying shear to the medium. 2. The method according to embodiment 1 , wherein a shear rate is applied in the range from about 90 sec-1 to 2829 sec-1 , preferably about 1100 sec-1 to 2200 sec-1 , or more preferably about 1500 sec-1 to 2000 sec-1.

[0108] 3. The method according to embodiment 1 or 2, wherein a shear stress is applied in the range from about T 10-2Pascal to about 1 ■ 10°Pascal.

[0109] 4. The method according to any one of the preceding embodiments, wherein the cell is selected from the group consisting of pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, somatic cell lines, and immortalized cell lines.

[0110] 5. The method according to any one of the preceding embodiments, wherein the cell is a nonhuman animal cell.

[0111] 6. The method according to embodiment 5, wherein the cell is a porcine or bovine cell.

[0112] 7. The method according to any one of the preceding claims, wherein the cell is a fat cell or a muscle cell or a precursor cell of either thereof.

[0113] 8. The method according to any one of the preceding embodiments, wherein the cells produced by the method are suitable for human and non-human dietary consumption.

[0114] 9. The method according to any one of the preceding embodiments, wherein the method is for the proliferation of the pluripotent cell.

[0115] 10. A cell or cell culture obtainable by or obtained by the method according to any of the preceding embodiments.

[0116] 11 . A cultured meat product for animal, preferably human, consumption, comprising at least one cell obtained by the method according to any one of embodiments 1-9, optionally further comprising a second cell type, such as (cultured) mammalian myocytes.

[0117] 12. A cultured fat or a cultured muscle product for animal, preferably human, consumption, comprising at least one cell obtainable by a method according to any one of embodiments 1-9.

[0118] 13. The method according to any one embodiments 1-9, further comprising the step of:

[0119] - incorporating the cultured cell into a food product for animal, preferably human, consumption. 14. Use of a cell obtained according to the method of any one of embodiments 1-9 or use of the method according to any one of embodiments 1-9 for tissue engineering, optionally for the production of cultured meat.

Claims

Claims1 . A method for reducing the size of stem cell aggregates during cell culture the method comprising culturing the pluripotent cells by perfusion in a cell culture medium and applying shear to the medium.

2. The method according to claim 1 , wherein a shear rate is applied in the range from about 90 sec-1 to 2829 sec-1 , preferably about 1100 sec-1 to 2200 sec-1 , or more preferably about 1500 sec-1 to 2000 sec-1.

3. The method according to claim 1 or 2, wherein a shear stress is applied in the range from about T 10-2Pascal to about 1 ■ 10°Pascal.

4. The method according to any one of the preceding claims, wherein the cell is selected from the group consisting of pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, somatic cell lines, and immortalized cell lines.

5. The method according to any one of the preceding claims, wherein the cell is a non-human animal cell.

6. The method according to claim 5, wherein the cell is a porcine or bovine cell.

7. The method according to any one of the preceding claims, wherein the cell is a fat cell or a muscle cell or a precursor cell of either thereof.

8. The method according to any one of the preceding claims, wherein the cells produced by the method are suitable for human and non-human dietary consumption.

9. The method according to any one of the preceding claims, wherein the method is for the proliferation of the pluripotent cell.

10. A cell or cell culture obtainable by or obtained by the method according to any of the preceding claims.

11. A cultured meat product for animal, preferably human, consumption, comprising at least one cell obtained by the method according to any one of claims 1-9, optionally further comprising a second cell type, such as (cultured) mammalian fat cells or (cultured) mammalian muscle cells or (cultured) precursor cells thereof.

12. A cultured fat or a cultured muscle product for animal, preferably human, consumption, comprising at least one cell obtainable by a method according to any one of claims 1-9.

13. The method according to any one claims 1-9, further comprising the step of: - incorporating the cultured cell into a food product for animal, preferably human, consumption.

14. Use of a cell obtained according to the method of any one of claims 1-9 or use of the method according to any one of claims 1-9 for tissue engineering, optionally forthe production of cultured meat.

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