Suspension culture of induced pluripotent stem cells using bioreactors

Abstract

The present invention provides a method for induced pluripotent stem cell (iPSC) culture through a reactor. The invention also provides an improved bioreactor which is particularly suitable for the culture of iPSC. The method and the bioreactor provided by the invention provide a universal technical platform for realizing large-scale commercial production of iPSC and differentiation of terminal functional cells.

Description

Technical Field

[0001] The present invention belongs to the field of stem cell technology, and specifically relates to a method for culturing induced pluripotent stem cells (iPSCs) through a reactor, providing a universal technical platform for realizing large-scale commercial production of iPSCs and differentiation of terminal functional cells. Background Art

[0002] In order to meet the needs of scientific research and clinical applications, the large-scale culture technology of induced pluripotent stem cells (iPSC) and their derivatives, that is, medium / large-scale culture using bioreactors, has also become the focus of many researchers at home and abroad. In previous attempts, there are many types of bioreactors involved in iPSC culture, including wall-attached, paddle-suspended, and paddle-free suspension. The wall-attached reactors mainly include reactors based on spherical carriers, sheet carriers, multi-plate reactors, and hollow fiber reactors. The cell surface cultured in these reactors has been greatly extended, but still relies on matrix gel. In the wall-attached culture system, the matrix gel and replacement fluid (cleaning fluid, culture medium, dissociation fluid or freezing fluid) are more lost, the dissociation efficiency of iPSC is not high, and its biggest problem is insufficient scalability, and its culture scale is still far from meeting the needs. Therefore, the recent methods at home and abroad are mainly to form self-assembled aggregates of iPSC in traditional stirred paddle reactors for suspension culture. In addition to the paddle-stirring type, the suspended reactor also includes the non-paddle-stirring type. However, because this type of reactor is mostly used in the currently more mature biomedical fields such as vaccines and antibodies, the cells involved are fully suspended single cells and have basically lost their wall adhesion, which is completely different from induced pluripotent stem cells. Therefore, the nutrient supply and waste liquid removal of the entire reaction system, oxygen supply, the frequency and method of support of culture medium and additives, the movement mode of the culture system, etc. may all be challenged.

[0003] Therefore, it is necessary to develop a stable, large-scale, suspension iPSC preparation system, for example, a closed and automated bioreactor, a finely regulated culture method, etc., to achieve clinical-grade or production-grade suspension culture of induced pluripotent stem cells. Summary of the invention

[0004] The present invention realizes continuous and closed production equipment and process for producing induced pluripotent stem cells (iPSCs). The present invention obtains iPSC aggregates by amplification through specific iPSC culture medium, specific reactor and appropriate culture parameters.

[0005] In the first aspect, the present invention provides a torrent bioreactor, comprising: a reactor tank; a cell culture bag fixedly mounted in the reactor tank, the cell culture bag comprising a flexible bag for accommodating cell culture, a liquid inlet line for injecting culture medium, a liquid outlet line for removing culture medium, a dedicated liquid exchange line and a retaining mechanism; and a driver connected to the reactor tank, the driver being configured to drive the reactor tank to move in a horizontal circular motion, thereby generating a torrent in the cell culture bag. In some preferred embodiments, the dedicated liquid exchange line comprises a first port extending outside the flexible bag body, a second port extending inside the flexible bag body, and a filter membrane attached to the second port, and the retaining structure is configured to enable the second port and the filter membrane to move from a first position to a second position, wherein in the first position, the second port and the filter membrane are located above the liquid surface of the culture medium and do not contact the culture medium, and in the second position, the second port and the filter membrane are located below the liquid surface of the culture medium. In a preferred embodiment, the second position is at the lower quarter to one-third of the height of the tank, i.e., from the bottom of the reactor tank to the quarter to one-third of the height of the tank.

[0006] In a specific embodiment, the holding mechanism includes a first magnet member located inside the flexible bag, a second magnet member located outside the flexible bag, and a fixing member for the first magnet member, wherein the first magnet member and the second magnet member are configured as follows: in the first position, the first magnet member and the second magnet member clamp the second port and the filter membrane to the top side wall of the flexible bag by magnetic force; and by manually removing the second magnet member, the magnetic force on the first magnet member is eliminated, thereby loosening the clamping of the second port and the filter membrane, and allowing them to move to the second position under the action of gravity. In a preferred embodiment, the first magnet member is a permanent magnet.

[0007] In a specific embodiment, the cell culture bag further comprises an inlet port and an exhaust port, wherein the inlet port is used to be in fluid communication with an external gas source. In a preferred embodiment, the exhaust port is in fluid communication with a pressure control valve such as a one-way valve located outside the cell culture bag, and an air filter is further provided between the exhaust port and the pressure control valve, and a heater is provided on the outside of the air filter for heating the gas discharged from the exhaust port.

[0008] In some specific embodiments, the rapid flow bioreactor further includes a temperature sensor, a pH sensor and a DO (dissolved oxygen) sensor inserted into the cell culture bag, wherein preferably, a flexible sleeve is provided on the outside of the temperature sensor so that the temperature sensor is not in direct contact with the culture medium. In some preferred embodiments, the rapid flow bioreactor further includes a temperature regulating module, a pH regulating module, a DO regulating module, and a gas flow regulating module. In some more preferred embodiments, the rapid flow bioreactor further includes a control unit, which receives signals from corresponding sensors and issues notifications and / or operates corresponding regulating modules to adjust corresponding indicators according to preset programs.

[0009] In some embodiments, there is provided use of the turbulent flow bioreactor of the present invention in cell culture, preferably, the cells are iPSCs.

[0010] In a second aspect, the present invention provides a stirred bioreactor, comprising: a cylindrical tank body, defining a cylindrical chamber for accommodating cell culture; an agitator extending into the cylindrical chamber; and a drive motor for driving the agitator to rotate; wherein the agitator has a stirring shaft, the first end of which is connected to the drive motor, and one or more coaxial stirring paddles are fixedly mounted on the shaft, as far as the second end of the stirring shaft. Each stirring paddle independently has the following structure: a cylindrical sleeve fixedly connected to the stirring shaft and having a common central axis with the stirring shaft, and three stirring blades evenly distributed along the outer circumference of the cylindrical sleeve, wherein each stirring blade has an angle of 45° with a plane perpendicular to the central axis, and the ratio of the diameter of the stirring blade to the diameter of the cylindrical tank body is 0.3 to 0.9, preferably 0.4 to 0.8; the diameter of the stirring blade is 2-5 cm, and more preferably the diameter of the stirring blade is 4 cm. The stirring paddle is a downward pressure type or an upward pressure type, preferably a downward pressure type.

[0011] In a specific embodiment, a stirring paddle is fixedly mounted on the stirring shaft, preferably substantially located at the second end of the shaft. In another specific embodiment, two coaxial stirring paddles are fixedly mounted on the stirring shaft, preferably one of which is substantially located at the second end of the shaft. In another specific embodiment, three or more coaxial stirring paddles are fixedly mounted on the stirring shaft, preferably one of which is substantially located at the second end of the shaft.

[0012] In a specific embodiment, the stirred bioreactor also includes a cover for enclosing the cylindrical chamber, an inlet pipe and an outlet pipe connected to the cylindrical chamber fluid through the cover, and an inlet port, an exhaust port and a sampling port arranged in the cover.

[0013] In some specific embodiments, the stirred bioreactor further comprises a temperature sensor, a pH sensor and a DO sensor inserted into the cylindrical chamber through the cover. In a preferred embodiment, a sleeve is provided on the outside of the temperature sensor so that the temperature sensor is not in direct contact with the culture medium, and the sleeve is a stainless steel sleeve or a flexible sleeve, preferably a stainless steel sleeve.

[0014] In some embodiments, the stirred bioreactor may further include an acid supply line, an alkali supply line, and a foam sensor.

[0015] In some embodiments, there is provided use of the stirred tank bioreactor of the present invention in cell culture, preferably, the cells are iPSCs.

[0016] In a third aspect, the present invention provides a method for culturing iPSCs, the method comprising inoculating iPSCs into a culture medium, and the method comprising using a bioreactor, characterized in that during the culture process, the culture medium containing iPSCs moves in a horizontal circle in the bioreactor, or mainly in a horizontal circle. The inventors accidentally discovered in the process of trying and analyzing various culture conditions that such a movement mode is quite conducive to the growth of iPSC aggregates into a suitable size and maintaining suspension, thereby showing rapid growth and expansion.

[0017] Therefore, in some embodiments, the iPSC culture method of the present invention comprises the steps of: inoculating iPSCs into a culture medium, wherein the culture medium is contained in a bioreactor; and causing the culture medium containing iPSCs to move in a horizontal circle, or mainly in a horizontal circle, in the bioreactor.

[0018] In a specific embodiment, the iPSC culture method of the present invention comprises the steps of:

[0019] (a1) adding a predetermined volume of culture medium into the bioreactor;

[0020] (a2) iPSCs are seeded into culture medium,

[0021] (a3) The culture medium containing iPSCs is moved in a horizontal circle, or mainly in a horizontal circle, in the bioreactor.

[0022] In a specific embodiment, the method further comprises a step (a0) before step (a1), wherein the iPSCs are cultured and expanded in a non-bioreactor manner for subsequent inoculation; preferably, the iPSCs are artificially cultured in a culture dish or culture bottle in an adherent or suspended manner to obtain at least about 2×10 7 Live cells for subsequent inoculation.

[0023] In some embodiments, the culture method of the iPSC of the present invention has a culture volume of at least 200mL. In some preferred embodiments, the culture method of the iPSC of the present invention has a culture volume of at least 300mL, at least 400mL, at least 500mL, at least 600mL, at least 700mL, at least 800mL, at least 900mL, at least 1000mL, at least 1100mL, at least 1200mL, at least 1300mL, at least 1400mL, at least 1500mL, at least 2000mL, at least 3000mL, at least 5000mL, at least 7500mL, at least 10000mL, at least 15000mL, at least 20000mL, at least 30000mL. In some preferred embodiments, the culture method of the iPSC of the present invention has a culture volume of 200mL-1500mL. In some preferred embodiments, the culture method of the iPSC of the present invention has a culture volume of 300mL-1500mL. In some more preferred embodiments, the culturing method of iPSCs of the present invention has a culture volume of 300 mL-600 mL.

[0024] In some embodiments, the bioreactor is a turbulent flow bioreactor, and its operating conditions include one or more of the following:

[0025] ① Monitor and maintain the reactor speed at 40-80rpm; ② Monitor and maintain the culture medium temperature at about 37°C; ③ Maintain the DO value of the culture system at 5-80%; ④ Maintain the pH between 6.8 and 7.4, and pass air when the pH value decreases or is too low, and pass CO2 gas when it increases or is too high; ⑤ Control the pressure in the bag to about 500Pa through the pressure control valve at the exhaust gas end, allow the bag to swell, and continuously heat the exhaust gas through the heater.

[0026] In some embodiments, the bioreactor is a stirred tank bioreactor and its operating conditions include one or more of the following:

[0027] ① Monitor and maintain the stirring paddle speed at 80-140rpm; ② Monitor and maintain the culture medium temperature at about 37°C; ③ The DO value of the culture system is maintained at 5-80%; ④ The pH is maintained between 6.8 and 7.4, and air is passed when the pH value decreases or is too low, and CO2 gas is passed when it increases or is too high; ⑤ The exhaust gas is discharged after being passed into the exhaust gas bottle.

[0028] In some more specific embodiments, air is passed through at a flow rate of 30 mL / min-80 mL / min in step (a1). In some more specific embodiments, air is passed through at a flow rate of 50 mL / min-100 mL / min in step (a2). In some more specific embodiments, air is passed through at a flow rate of 100 mL / min-300 mL / min in step (a3).

[0029] In some embodiments, in step (a2), the cell density in the bioreactor after inoculation is 1.5×10 5 Viable cells / mL and no more than 3.5×10 5 Viable cells / mL.

[0030] In some embodiments, the step (a2) further comprises adding an additive to the culture system; in some further embodiments, the additive comprises ROCKi, such as Y-27632. In some further embodiments, the additive further comprises a growth factor, such as bFGF.

[0031] In some embodiments, the culture medium is hPSC-AOF, hPSC-Scale, or Essential 8.

[0032] In some preferred embodiments of the iPSC culture method of the present invention, the iPSCs grow in a manner of forming aggregates.

[0033] In some embodiments, the step (a3) ​​comprises monitoring and maintaining the reactor operating speed at 40 to 80 rpm while the bioreactor is running; in some embodiments, the step (a3) ​​comprises monitoring and maintaining the stirring paddle operating speed at 80 to 140 rpm while the bioreactor is running; in some embodiments, the step (a3) ​​comprises monitoring and maintaining the culture medium temperature at approximately 37°C while the bioreactor is running.

[0034] In some embodiments, step (a3) ​​includes monitoring and maintaining the DO value of the culture system at 5-80% and the pH at between 6.8 and 7.4 during the operation of the bioreactor. To this end, air is continuously passed at a flow rate of 100 mL / min-300 mL / min. Air is passed when the pH value decreases or is too low, and CO2 gas is passed when the pH value increases or is too high.

[0035] In some embodiments, the step (a3) ​​also includes a fluid replacement operation once a day. Preferably, the fluid replacement operation replaces about 25% to about 75% of the volume of the culture medium in the culture system. More preferably, the fluid replacement operation replaces about 50% of the volume of the culture medium in the culture system. In a preferred embodiment of the present invention, the fluid replacement operation is not performed before most of the cultured iPSCs grow in aggregates greater than 100 μm. In some more specific embodiments, the fluid replacement operation includes the use of a cell filter bag externally connected to a bioreactor, preferably the bioreactor is a stirred bioreactor. In some more specific embodiments, the fluid replacement operation includes the use of a single dedicated fluid replacement line with a cell filter membrane. In some more specific embodiments, the fluid replacement operation includes making the inlet and outlet lines independent of each other.

[0036] In some embodiments, the fluid exchange operation includes exchanging cell fluid through a dedicated fluid exchange pipeline; preferably, the cell fluid exchange includes the following steps: maintaining or preferably appropriately increasing the reactor speed; replacing 25% to 75%, preferably about 50% of the volume of the culture system through the fluid exchange pipeline. The culture medium volume ratio; after running at the increased reactor speed for at least 1-5 minutes, gradually adjusting back to the original speed.

[0037] In some embodiments, the fluid replacement operation includes performing cell fluid replacement through an external cell filter bag; preferably, the cell fluid replacement includes the following steps: pausing the operation of the reactor; extracting the culture fluid into an external cell filter bag, retaining the aggregates by filtration and removing 25% to 75%, preferably about 50% of the volume of the culture system, and then adding a roughly equal volume of fresh culture medium, and finally returning the culture that has completed the fluid replacement to the tank and resuming the operation of the reactor.

[0038] In some embodiments, the fluid replacement operation includes using a dual inlet / outlet pipeline as a fluid replacement channel. Preferably, the cell fluid replacement includes the following steps: at least about 5 minutes before the start of the fluid replacement, pausing the movement of the bioreactor to allow the aggregates in the culture medium to settle in the lower half or even the bottom of the culture system; then completing the fluid replacement by extracting the old culture medium in the upper half of the culture system and adding new culture medium, and the fluid replacement volume is 25% to 75% of the volume of the culture system, preferably about 50%.

[0039] In some embodiments, the bioreactor is a turbulent bioreactor, and the step (a3) ​​includes controlling the pressure in the bag to about 500 to 1500 Pa through a pressure control valve at the tail gas end when the bioreactor is running, so that the bag is bulged. In some embodiments, the bioreactor is a turbulent bioreactor, and the step (a3) ​​includes continuously heating the tail gas through a heater, and the tail gas is uniformly collected and processed after passing into a tail gas bottle.

[0040] In a preferred embodiment, the iPSC culture method of the present invention is carried out in a bioreactor of the present invention. In a preferred embodiment, the iPSC culture method of the present invention is carried out in a torrent bioreactor of the present invention. In a preferred embodiment, the iPSC culture method of the present invention is carried out in a stirred bioreactor of the present invention.

[0041] In some embodiments, the iPSC culture method of the present invention further comprises the step of harvesting the cultured iPSCs. In some embodiments, when the iPSCs in the culture system reach about 1 to 2×10 6 The cultured iPSCs were harvested at a cell density of 1.5 viable cells / mL.

[0042] In some embodiments, the harvesting step includes collecting all of the aggregate suspension (ie, culture medium containing aggregates) into a receiving flask connected to the bioreactor, followed by dissociation of the aggregates into single cells.

[0043] In some embodiments, the harvesting step includes: collecting all the aggregate suspensions into a receiving bottle connected to the bioreactor, transferring a certain volume of the aggregate suspension to a centrifuge tube for dissociation, maintaining horizontal circular motion of the receiving bottle for about 1 to 3 minutes before transferring the suspension to evenly suspend all the aggregates in the bottle; discarding the supernatant, adding DPBS, discarding the supernatant after settling for 1 to 10 minutes, adding 2 to 4 times diluted dissociation solution, dissociating for 5 to 20 minutes, dissociating the aggregates into a homogeneous single cell suspension, adding culture medium to terminate the reaction, and harvesting the cells. In some preferred embodiments, the dissociation solution includes Accutase, Accumax and NB6, Gentle Cell Dissociation Reagent, etc.

[0044] In some embodiments, the iPSC culture method of the present invention can achieve at least 5-fold doubling within the culture period of one passage, such as 5-fold doubling / 4-6 days (eg, 4 days). In some embodiments, the culturing method of iPSCs of the present invention can achieve a doubling of at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 17 times, or at least 20 times within the culture period, such as at least 6 times / 4-6 days (e.g., 4 days), at least 7 times / 4-6 days (e.g., 4 days), at least 8 times / 4-6 days (e.g., 4 days), at least 9 times / 4-6 days (e.g., 4 days), at least 10 times / 4-6 days (e.g., 4 days), at least 11 times / 4-6 days (e.g., 4 days), at least 12 times / 4-6 days (e.g., 4 days), at least 13 times / 4-6 days (e.g., 4 days), at least 14 times / 4-6 days (e.g., 4 days), at least 15 times / 4-6 days (e.g., 4 days), at least 17 times / 4-6 days (e.g., 4 days), or at least 20 times / 4-6 days (e.g., 4 days).

[0045] In some embodiments, the advantages of the iPSC culture method of the present invention include no need for matrix gel, high degree of automation, simple process, small batch-to-batch difference, complete and traceable process data, and little effect on cell growth state after optimization or removal of the reactor stirring paddle.

[0046] In some embodiments, the iPSC culture method of the present invention further comprises a quality control / quality inspection step for the harvested cells. Preferably, the quality control / quality inspection results show that the pluripotency of the cells is substantially consistent with that of manual culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Combined with the following Figure 1 When reading together, the preferred embodiments of the present invention described in detail below will be better understood. For the purpose of illustrating the present invention, the drawings show the currently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangement and means of the embodiments shown in the drawings.

[0048] Figure 1 A schematic structural diagram of an embodiment of a torrent-type bioreactor of the present invention is shown, wherein the reactor vessel is cut away to show the cell culture bag and corresponding components located therein.

[0049] Figure 2 A schematic structural diagram of an embodiment of a cell culture bag of the present invention is shown.

[0050] Figure 3 Shows Figure 1 A top view of the turbulent flow bioreactor is shown.

[0051] Figure 4 A schematic diagram of the structure of a single stirring blade example of a stirred bioreactor of the present invention is shown.

[0052] Figure 5 A schematic structural diagram of an example of a stirred bioreactor with multiple stirring blades according to the present invention is shown.

[0053] Figure 6 Shows Figure 4 Or a top view of a stirred bioreactor as shown in FIG5 .

[0054] Figure 7 An example of a stirring blade of the present invention is shown, and 7A is a perspective view thereof, and 7B is a top view thereof.

[0055] Figure 8 The graph shows the growth of iPSC aggregates cultured in a torrent bioreactor according to the present invention, and the control group is cultured in a shake flask.

[0056] Fig. 9 The particle size analysis curve of iPSC aggregates cultured in the torrent bioreactor of the present invention is shown, and the control group is cultured in a shake flask.

[0057] Fig.10 The figure shows the result of detecting pluripotency markers by qPCR experiment in iPSC aggregates cultured in the torrent bioreactor of the present invention.

[0058] Fig.11 The figure shows the result of detecting pluripotency markers by FACS experiment in iPSC aggregates cultured in the torrent bioreactor of the present invention.

[0059] Fig.12 The graph shows the growth of iPSC aggregates cultured in a stirred bioreactor of the present invention, and the control group is cultured in a shake flask.

[0060] Fig.13 A particle size analysis curve of iPSC aggregates cultured in a stirred bioreactor of the present invention is shown.

[0061] Fig.14 The figure shows the result of detecting pluripotency markers by FACS experiment in iPSC aggregates cultured in the stirred bioreactor of the present invention.

[0062] Fig.15 Shown is a picture of iPSC aggregates growing in a horizontal agitator bioreactor.

[0063] Fig.16 A graph showing the results of qPCR experiments to detect pluripotency markers in iPSC aggregates cultured in a horizontal agitator bioreactor.

[0064] Fig.17Shown is a picture of iPSC aggregate growth cultured in a wave-type bioreactor.

[0065] Fig.18 A graph showing the results of qPCR experiments to detect pluripotency markers in iPSC aggregates cultured in a wave-type bioreactor.

[0066] Fig.19 A graph showing the analysis of proliferation results of iNK differentiation from iPSC aggregates using the stirred bioreactor of the present invention.

[0067] Fig. 20 The figure shows the FACS experimental results of iNK differentiation of iPSC aggregates using the stirred bioreactor of the present invention.

[0068] Fig.21 A diagram showing the reactor tank and impeller design for a horizontal stirred-paddle bioreactor.

[0069] Fig. 22 A perspective view of the wave bioreactor and a reaction bag culture display showing the process of culturing iPSC aggregates are shown. DETAILED DESCRIPTION OF THE INVENTION

[0071] definition

[0072] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

[0073] The term "about" when used in conjunction with a numerical value is intended to encompass numerical values ​​within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value. For example, "about 50%" means all numerical values ​​within the range of 47.5%-52.5%.

[0074] When used in conjunction with two or more alternatives, the expression "and / or" should be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.

[0075] As used herein, the term "comprising" or "including" means including the mentioned elements, integers or steps, but does not exclude any other elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise specified, the situation consisting of the mentioned elements, integers or steps is also covered.

[0076] As used herein, "expression markers" or "markers" can be used to determine the identity of a cell type. Certain informative DNA sequences of cell-specific genes can be transcribed into mRNA and are usually subsequently translated into proteins (gene products) that perform specific functions in cells. The expression of such markers can be detected and quantified at the RNA level or protein level by methods known in the art to characterize the cells. Markers that can be used to characterize pluripotent stem cells and three germ layer (mesoderm, endoderm and ectoderm) cell lineages are well known in the art.

[0077] iPSCs and their culture

[0078] In this article, the term "pluripotent stem cell" (PSC) refers to an undifferentiated cell with differentiation potential and proliferation ability (particularly self-renewal ability) and maintaining differentiation potential. According to the differentiation potential, stem cells include subgroups such as PSC, multipotent stem cells, and unipotent stem cells. PSC refers to a stem cell that can be cultured in vitro and has the potential to differentiate into any cell lineage belonging to the three germ layers (ectoderm, mesoderm, endoderm). In this article, the term "pluripotency" or "stemness" refers to the ability of cells to produce progeny and to differentiate into cell types of the three germ layers (endoderm, mesoderm, and ectoderm) under appropriate conditions. Standard test methods recognized in the art can be used, for example, the ability to form teratomas in 8-12 week-old SCID mice, or the three-lineage differentiation method described in the embodiment, to establish the pluripotency of a cell population. It is also possible to detect pluripotent cells by identifying other pluripotent stem cell characteristics. The characteristics include, but are not limited to, expressing or not expressing certain molecular marker combinations.

[0079] PSC can be induced from fertilized eggs, cloned embryos, germ stem cells, stem cells in tissues, somatic cells, etc. Examples of PSC include embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), etc. In this article, induced pluripotent stem cells (also referred to as "iPSC" in this article) refer to cells that are transformed from somatic cells by expressing or inducing the expression of a specific combination of pluripotency-related genes, and have the ability to differentiate into various cell types that show characteristics related to the cell lineages of three germ layers (endoderm, mesoderm and ectoderm). Typically, iPSC (i.e., iPS cells) have typical pluripotent stem cell morphology, can self-renew, have tri-lineage differentiation ability, and express a combination of multiple pluripotency markers (e.g., OCT4, NANOG and TRA-1-81). In this article, the bioreactor and culture method according to the present invention are particularly suitable for the culture of iPSC, but as those skilled in the art understand, the method of the present invention may also be applicable to the identification / screening of other PSC or PSC-derived cell populations. Therefore, herein, when referring to "iPSC", it should be understood that the expression can also be replaced by "PSC", although preferably according to the embodiments of the present invention, the iPSC refers to induced pluripotent stem cells.

[0080] The cell markers of human pluripotent stem cells can be selected from one or more of the following: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, ALP, PODXL, SOX2, E-cadherin, EPCAM, KLF4, LIN-28A, SOX11, SALL2, PRDM14, OCT4 (POU5F1), REX1 and NANOG. The cell morphology associated with pluripotent stem cells is also a feature of pluripotent stem cells. Typically, on a two-dimensional culture matrix, pluripotent stem cells typically grow as flat colonies with clear edges, and cells grow densely in the colonies. Under a phase contrast microscope, the morphology of pluripotent cells can be distinguished from the morphology of somatic cells, such as fibroblasts have a characteristic long spindle shape and the cell volume is significantly larger.

[0081] Generally speaking, iPSCs are anchorage-dependent cells, but they can also be cultured in suspension in the form of aggregates. Therefore, iPSCs can be cultured using two different culture systems: anchorage-dependent culture and suspension culture. iPSC suspension culture is easier to achieve large-scale commercial production than static adherent culture.

[0082] As used herein, "adherent" means that the cells adhere to a vessel, for example, in the presence of an appropriate culture medium, the cells adhere to a sterile plastic (plastic-coated) cell culture dish or flask.

[0083] In this article, "suspension culture" refers to such a culture system or culture method: cells, single cells, cell collections, or a mixture of single cells and collections therein are suspended in a culture medium rather than adhering to a surface. In a liquid culture medium, under constant stirring, the cultured cells are amplified in a non-adherent manner and grow in a three-dimensional direction in the form of a single cell or a small cell mass. Especially for the latter, although actually in suspension, the mutual adhesion of cells in the cell mass also provides a state similar to "adherence", and there is also an extracellular matrix (ECM) between the cells in the cell mass, which meets the needs of iPSC. Such cell clusters can be referred to as "aggregates", "aggregates" or "aggregates", etc. Pluripotent stem cells can be cultured under dynamic conditions (i.e., under conditions where pluripotent stem cells are subjected to continuous motion during suspension culture, such as a stirred suspension culture system), while retaining their proliferation ability, pluripotency ability, and chromosome karyotype stability in multiple passages.

[0084] Obviously, the distribution of nutrients and oxygen in the aggregates is limited and uneven. In addition, due to various factors such as the change of ECM by cell growth, only aggregates of a certain size have the best growth conditions and proliferation rate. Therefore, in the iPSC suspension culture process, the culture state of the aggregates must be controlled. The ideal solution is to produce many aggregates of controllable size, and to have sufficient contact between the cells so that they can grow stably. On the one hand, the aggregates are easy to precipitate and accumulate at the bottom of the bioreactor tank (i.e., the container part that holds the culture), so it is necessary to drive the culture fluid to move (e.g., stirring) to prevent the above problems. On the other hand, during the suspension culture process, mechanical force (e.g., stirring) is applied to drive the culture fluid to flow, and the cells will be subjected to the shear stress generated by the flow, which also affects the survival and differentiation of iPSC. Therefore, the optimal driving level that can prevent sedimentation in the bioreactor while also preventing cells from being damaged by shear force is very important for the successful suspension culture of the bioreactor.

[0085] Shear rate and shear stress are mechanical characteristics that define the shear of the fluid in the system. The rotation rate of the culture medium in the bioreactor and the diameter of the aggregates are related to the shear rate and shear stress in the fluid. The inventors found that when the culture medium containing iPSCs of appropriate density is moved in a circular manner at a certain rate in the bioreactor, the shear stress is sufficient to make aggregates of a certain size float in the culture medium and maintain the appropriate size without merging with each other to produce overly large aggregates, and the cells will not be damaged or even broken by the shear stress.

[0086] Therefore, the present invention provides a method for culturing iPSCs, the method comprising inoculating iPSCs into a culture medium, and the method comprising using a bioreactor, characterized in that during the culturing process, the culture medium containing iPSCs moves in a horizontal circle in the bioreactor, or mainly in a horizontal circle. In a specific embodiment, the method for culturing iPSCs of the present invention comprises the steps of: (a1) adding a culture medium to a bioreactor according to a predetermined volume; (a2) inoculating iPSCs into the culture medium; (a3) ​​causing the culture medium containing iPSCs to move in a horizontal circle in the bioreactor, or mainly in a horizontal circle.

[0087] As mentioned above, iPSC can grow and proliferate in the form of "aggregates" in suspension culture. The term "aggregate" herein, also known as "spheroid", "aggregate" or "aggregate", means the phenomenon that dissociated cells mixed in vitro tend to group with other cells, also known as "floating aggregates". Compared with common single-cell suspensions, cell aggregates contain dozens or even thousands of cells, which are connected by connecting adhesion and extracellular matrix (ECM) that jointly contribute to the final differentiation product. In this regard, cell aggregates can be defined as a type of tissue that will have certain performance advantages under specific culture conditions.

[0088] In certain preferred embodiments of the present invention, the average aggregate diameter is from about 30 μm to about 450 μm, from about 30 μm to about 400 μm, or from about 30 μm to about 350 μm. According to some embodiments, the average aggregate diameter is greater than 150 μm. According to certain exemplary embodiments, the average aggregate diameter is from about 70 μm to about 320 μm. According to certain exemplary embodiments, the average aggregate diameter is from about 80 μm to about 200 μm. According to certain exemplary embodiments, the average aggregate diameter is from about 100 μm to about 300 μm.

[0089] The present invention provides a method for culturing iPSCs in suspension, wherein an iPSC single cell suspension produces iPSC aggregates. Without wishing to be bound by theory, a variety of mechanical and non-physiological factors affect the movement and aggregation of cultured cells, for example, factors such as the fluid mechanical microenvironment such as shear rate and shear stress, the movement patterns of cells in culture vessels of different shapes and sizes, different culture medium conditions, and cell density and the concentration of multiple growth factors in the culture medium are all related to optimal cell aggregate viability and performance.

[0090] Preferably, in order to suspend and culture iPSC, a control culture system (preferably a computer-controlled culture system) is used to achieve culture according to some embodiments of the present invention, and a suitable device is used in the control culture system to automatically monitor and control culture parameters, such as temperature, rotation speed, pH and DO. Once the desired culture parameters are determined, the system can be configured to automatically adjust the culture parameters as needed to enhance pluripotent stem cell expansion and differentiation. The terms "dissolved oxygen" and "DO" of the present invention are respectively the Chinese and English abbreviations of the same concept, and are used interchangeably in the present invention. The term refers to the amount of dissolved oxygen in an aqueous solution (e.g., culture medium). Because under culture conditions, the oxygen content in the air and the temperature of the culture medium are relatively constant, how to increase the dissolved oxygen content of the culture medium by adjusting indicators such as ventilation and movement patterns is an important task of the culture system.

[0091] In some embodiments of the present invention, in order to avoid excessive changes in the pH value and ion environment of the culture environment that may cause cell growth inhibition, the pH value is controlled by introducing different gases into the culture medium rather than directly adding acidic or alkaline reagents.

[0092] Culture medium

[0093] iPSCs can be cultured in any medium that provides sufficient nutrients and environmental stimuli to promote growth and expansion.

[0094] At present, the culture medium for iPSC culture has become a commonly purchased and used reagent in the field. The preferred basal culture medium is a chemically defined culture medium that has no animal-derived components and almost completely uses human recombinant proteins and organic compounds. Commonly used products of this type can be included in the technical solution of the present invention. The preferred basal culture medium is hPSC-Scale, hPSC-AOF or Essential 8.

[0095] In addition, the iPSC culture system can and includes one or more additives, such as, but not limited to, ROCK (Rho-kinase) inhibitors (ROCKi), growth factors such as basic fibroblast growth factor (bFGF). In some embodiments, ROCKi is selected from: Thiazovivin, Fasudil, Ripasudil, Netarsudil, RKI-1447, Y-27632, GSK429286A, Y30141, etc., or any combination of at least two of the above. The concentration of ROCKi may be about 1 to about 100 μM, about 1 to 90 μM, about 1 to about 80 μM, about 1 to about 70 μM, about 1 to about 60 μM, about 1 to about 50 μM, about 1 to about 40 μM, about 1 to about 30 μM, about 1 to about 20 μM, about 1 to about 15 μM, about 1 to about 10 μM, or about 10 μM. In one embodiment, at least 1 μM, at least 5 μM or at least 10 μM ROCKi is added. In one embodiment, 10 μM ROCKi is added.

[0096] Fluid exchange

[0097] Commercial-scale mammalian cell culture processes can be carried out in a variety of different operating modes, such as batch mode, fed-batch mode, perfusion mode, semi-continuous mode, and the like. Most commercial-scale mammalian cell culture processes are operated in a fed-batch mode, in which the old culture medium containing the cells is removed and new culture medium and nutrient factors are added during the feeding process. This operation facilitates the replenishment of nutrients to support further growth and reproduction of cells, reduces the number of cells so that the cell density is controlled within a reasonable range, and reduces the concentration of metabolites to eliminate the inhibition of excessive metabolites on the cell growth process. In a preferred embodiment of the present invention, the culture mode is a fed-batch-like mode: fresh culture medium is added to the culture system at multiple time points at certain time intervals, but because the number and density of iPSCs have not yet reached the time of harvesting, the removal of the old culture medium is almost not accompanied by the harvesting of the cultured cells.

[0098] The fluid replacement operation of the present invention is based on the stringent requirements of iPSC for culture conditions as mentioned above. While ensuring the culture density, it is necessary to ensure that the amount of growth-promoting substances such as nutrients and growth factors in the culture medium is sufficient, while the concentration of metabolites is maintained at a low level to avoid their inhibition of growth. Therefore, the time interval for fluid replacement cannot be too long or too short.

[0099] In some embodiments of the present invention, the step (a3) ​​includes a fluid change operation. In some embodiments of the present invention, calculated from the cell inoculation time day0, from day1 to day4 or day5, the fluid is changed once a day. In some specific embodiments of the present invention, the time point for the first fluid change is 18-36h after inoculation, such as 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h. In some specific embodiments of the present invention, the time interval between two fluid changes is 18-36h, such as 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h.

[0100] In some preferred embodiments of the present invention, the fluid replacement operation replaces about 25% to about 75% of the volume of the culture medium in the culture system. More preferably, the fluid replacement operation replaces about 50% of the volume of the culture medium in the culture system. In a preferred embodiment of the present invention, the fluid replacement operation is not performed before most of the cultured iPSCs grow in aggregates greater than 100 μm. In some more specific embodiments, the fluid replacement operation includes the use of a cell filter bag externally connected to a bioreactor, preferably the bioreactor is a stirred bioreactor. In some more specific embodiments, the fluid replacement operation includes the use of a single dedicated fluid replacement line with a cell filter membrane. In some more specific embodiments, the fluid replacement operation includes making the inlet and outlet lines independent of each other.

[0101] Traditional cultivation method

[0102] For iPSC, in the earliest traditional adhesion culture, cells were seeded on a matrix containing feeder cells (such as mouse embryonic fibroblasts), which can lead to cross-contamination of cells. Therefore, as an improvement, various extracellular matrix (ECM) coatings have been developed to replace the use of feeder cells, such as matrigel and recombinant laminin or vitronectin. Alternatively, iPSC suspension culture can also be performed in a shake flask, such as a series of culture flasks with increasing volume. Relatively small bioreactors can also be used to culture iPSC inoculum.

[0103] In a specific embodiment, the method further comprises a step (a0) before step (a1), wherein the iPSCs are cultured and expanded in a non-bioreactor manner for subsequent inoculation; preferably, the iPSCs are artificially cultured in a culture dish or culture bottle in an adherent or suspended manner to obtain, for example, at least about 2×10 7 Live cells for subsequent inoculation.

[0104] For iPSC non-dynamic culture, pluripotent stem cells can be cultured in the following containers: culture dishes, flasks, shake flasks, or coated or uncoated cell factories, etc.

[0105] Suspension culture bioreactor

[0106] Stirred Bioreactor

[0107] The paddle type of suspension culture reactor is divided into horizontal paddle type and vertical wheel paddle type according to the stirring axis, and the non-paddle type is divided into wave type and torrent type according to the movement mode. In the bioreactor, the direction of liquid movement is mainly circumferential, radial and axial.

[0108] The terms "paddle bioreactor" and "stirred bioreactor" are used interchangeably herein, and have at least a cylindrical chamber for accommodating culture medium and one or more groups of stirring paddles immersed in the culture medium and driven to rotate by a drive outside the chamber. For a paddle bioreactor, the blades have a dual role. On the one hand, the rotating blades can drive the liquid in the reactor to move, thereby achieving mixing of substances in the liquid, which is beneficial to the absorption of nutrients by cells in the entire reactor. On the other hand, the rotation of the blades causes shear stress, which can cause cell damage when it reaches a certain value. The structure and rotation speed of the blades determine the mixing effect and the magnitude of the shear force generated in the reactor. These two factors are often contradictory, that is, the shear force generated when the mixing effect is good is large, and the shear force generated when the mixing effect is poor is small. How to minimize the shear force generated by stirring while ensuring the mixing effect is the key to the design of bioreactor blades.

[0109] The inventor has proposed an optimized blade through multiple calculations and attempts, which can produce a liquid flow dominated by circumferential flow in the culture medium, and mix a certain radial flow and axial flow. This combined flow can achieve a good mixing effect, and the shear force generated is also small, which is suitable for the suspended growth of iPSC aggregates. The stirring paddle is a three-blade, spiral blade, the angle between the blade and the plane of the vertical rotation axis is 45°, and the projection of each blade on the plane is a fan-shaped shape of approximately 120°. The ratio of the diameter of the stirring blade to the diameter of the cylindrical chamber in which it is located is 0.3 to 0.9, preferably 0.4 to 0.8. During operation, the blade pushes the liquid flow on the motion path downward or upward, preferably downward. Although in the local part of the blade, the liquid presents an axial flow from top to bottom or from bottom to top driven by the blade, but in general, due to the setting of the shape and size of the blade, and its specific ratio with the chamber, the stirring paddle of the present invention applies circumferential circular motion and overall homogenization to the culture medium in the reactor tank, increasing the overall volume flow of the fluid.

[0110] Therefore, the present invention also provides a stirred bioreactor, comprising: a cylindrical tank body, defining a cylindrical chamber for accommodating cell culture; an agitator extending into the cylindrical chamber; and a drive motor for driving the agitator to rotate; wherein the agitator has a stirring shaft, the first end of which is connected to the drive motor, and one or more coaxial stirring paddles are fixedly mounted on the shaft, as far as the second end of the stirring shaft. Each stirring paddle independently has the following structure: a cylindrical sleeve fixedly connected to the stirring shaft and having a common central axis with the stirring shaft, and three stirring blades evenly distributed along the outer circumference of the cylindrical sleeve, wherein each stirring blade has an angle of 45° with a plane perpendicular to the central axis, the ratio of the diameter of the stirring blade to the diameter of the cylindrical tank body is 0.3 to 0.9, preferably 0.4 to 0.8, and the diameter of the stirring blade is 2-5 cm, more preferably 4 cm.

[0111] In a specific embodiment, the stirred bioreactor also includes a cover for enclosing the cylindrical chamber, an inlet pipe and an outlet pipe connected to the cylindrical chamber fluid through the cover, and an inlet port, an exhaust port and a sampling port arranged in the cover.

[0112] In some specific embodiments, the stirred bioreactor further comprises a temperature sensor, a pH sensor and a DO sensor inserted into the cylindrical chamber through the cover. In a preferred embodiment, a sleeve is provided on the outside of the temperature sensor so that the temperature sensor is not in direct contact with the culture medium, and the sleeve is a stainless steel sleeve or a flexible sleeve, preferably a stainless steel sleeve.

[0113] In some embodiments, the stirred bioreactor may further include an acid supply line, an alkali supply line, and a foam sensor.

[0114] Those skilled in the art will appreciate that, for stirred bioreactors, the stirring blades have a dual role. On the one hand, the rotating stirring blades can drive the liquid in the reactor to move, thereby achieving mixing of the substances in the liquid, which is beneficial for the cells in the entire reactor to absorb nutrients. On the other hand, the rotation of the stirring blades causes shear stress, which can cause cell damage when it reaches a certain value. In most stirred bioreactors, the speed of the stirring blades is tens to thousands of revolutions per minute (rpm). Generally speaking, the power of the driving motor does not constitute a limitation, so the speed of the stirring blades is calculated and set by the operator according to the various fluid mechanics indicators of the culture system and the desired effect.

[0115] The improved stirring paddle of the present invention can minimize the shear force generated by the stirring paddle while ensuring the mixing effect at a specific rotation speed, such as 80-140 rpm. Therefore, in a preferred embodiment, the rotation speed of the stirring paddle is 80-140 rpm.

[0116] In the bioreactor of the present invention, the design of the impeller blades provides rapid mixing, maintains homogeneity, maintains cell suspension and bubble dispersion, and minimizes cell damage caused by shear forces. The bioreactor of the present invention provides a physicochemical environment that is homogeneous in terms of DO, pH and temperature, nutrient and metabolite concentration gradients.

[0117] In other aspects, like the stirred bioreactors known in the art, the stirred bioreactor of the present invention may also include sensors / probes and one or more connections (e.g., tubes, valve holes) for fluids and gases, etc. to enter the cell culture chamber of the bioreactor, and may also include one or more pumps, controllers and electronic devices (e.g., heating devices, cooling devices, acid reagent sources, alkaline reagent sources, defoaming agent sources, pressurization / pressure relief devices, sensor electronics, pressurized gas controllers, liquid (e.g., culture medium) sources, etc.). Important processing parameters monitored and controlled by the sensors / probes and controllers include any one or more combinations of temperature, movement pattern and speed, pH, DO, gas flow rate (for example), etc. Those skilled in the art are capable of making routine adjustments to known components and control strategies within the scope of known stirred bioreactors according to their functions.

[0118] In a preferred embodiment, the iPSC culture method of the present invention is carried out in the stirred bioreactor of the present invention.

[0119] Fluid flow bioreactor

[0120] In addition, in recent years, the use of reactors without stirring blades can reduce the damage to cells during the culture process, and relevant quality control data show that the pluripotency of cells is basically the same as that of manual culture. Cell culture is carried out in disposable consumables, which has low requirements for the operating environment, low contamination rate, and lower labor and material costs for harvesting the same number of cells.

[0121] Animal cells are sensitive to shear forces due to the lack of cell walls. The cells are fragile, resulting in low survival rates, and the demand for dissolved oxygen increases during high-density and large-scale culture. However, traditional bioreactors (such as wave reactors and horizontal stirred paddle bioreactors) increase the dissolved oxygen content by improving the oxygen mass transfer efficiency during high-density and large-scale culture of animal cells, and the way to improve the oxygen mass transfer efficiency is to increase the rotation speed or increase the deep oxygen ventilation volume. However, both methods will bring a large amount of foam and higher shear forces, thereby causing more cell damage, and reduce the carbon dioxide dissipation efficiency in the cell fluid, making it difficult for cells to maintain normal cell proliferation and resulting in a decrease in cell survival rate, and then it is difficult to achieve high-density and large-scale culture of animal cells, especially animal cells that are more sensitive to shear forces.

[0122] The torrent bioreactor is a new type of non-bubbling bioreactor that has emerged in recent years. Its tank body generally has a cylindrical tank body and a conical tank bottom. The principle of this type of bioreactor is that the tank body rotates at a set rate around a given central axis (generally the central axis of the base platform) (for example, revolution, that is, the reactor tank body rotates horizontally in a circle), so that the liquid in the tank body generates torrents with the rotational inertia, forming a local film on the reactor wall. Due to the high gas-liquid mass transfer efficiency of the film, it is quickly saturated with oxygen in the top space and then combined with the liquid body. This process is repeated, continuously providing extremely strong oxygen mass transfer efficiency in the local film, thereby ensuring the dissolved oxygen content of the culture medium in the entire culture system. As described, the torrent bioreactor does not contain a stirring and foaming device, so the shear force generated on the cells is very low. In addition, the gas-liquid mass transfer efficiency is high, the parameter control is precise, the experimental operation is simple, it is easy to use disposable culture bags, and the shortcomings of wave reactors and stirred reactors can be cleverly avoided. It is a better reactor for animal cell (e.g., iPSC) culture. At the same time, the rapid flow bioreactor also has good scalability.

[0123] As conventionally provided in the art, the bioreactor, as necessary for achieving the purpose of cell culture, will include a tank body for containing the culture system. Most bioreactors may also include liquid inlet / outlet pipelines and air inlet / exhaust pipelines, as well as corresponding spare pipelines. In order to monitor and regulate the cell culture conditions in real time, online monitoring components such as temperature sensors, pH sensors, DO sensors, and heating / cooling elements, acid / base supplementation elements / pipelines, etc. may also be provided. Preferably, the bioreactor may also include a control unit to realize automatic / semi-automatic monitoring of various indicators of the culture system, and automatically adjust according to the monitoring results, and / or notify manual intervention when necessary.

[0124] The present invention provides an improved bioreactor by improving and optimizing an existing bioreactor to make it more suitable for the culture of iPSCs, preferably more suitable for the culture of iPSCs using the method of the present invention.

[0125] In some embodiments of the present invention, the present invention provides a torrent-type bioreactor, comprising: a reactor tank; a cell culture bag fixedly mounted in the reactor tank, the cell culture bag comprising a flexible bag for accommodating cell culture, a liquid inlet line for injecting culture medium, a liquid outlet line for removing culture medium, a dedicated liquid exchange line and a retaining mechanism; and a driver connected to the reactor tank, the driver being configured to drive the reactor tank to rotate horizontally, thereby generating torrents in the cell culture bag. In some preferred embodiments, the dedicated liquid exchange line comprises a first port extending outside the flexible bag body, a second port extending inside the flexible bag body, and a filter membrane attached to the second port, and the retaining structure is configured to enable the second port and the filter membrane to move from a first position to a second position, wherein in the first position, the second port and the filter membrane are located above the liquid surface of the culture medium and do not contact the culture medium, and in the second position, the second port and the filter membrane are located below the liquid surface of the culture medium. In a preferred embodiment, the second position is between the lower quarter to one-third height of the tank, that is, between the bottom of the reactor tank and the quarter to one-third height of the tank.

[0126] In a specific embodiment, the retaining mechanism includes a first magnet member located inside the flexible bag body, a second magnet member located outside the flexible bag body, and a fixing member for the first magnet member, wherein the first magnet member and the second magnet member are configured as follows: in the first position, the first magnet member and the second magnet member clamp the second port and the filter membrane to the top side wall of the flexible bag body through magnetic force; and by manually removing the second magnet member, the magnetic force on the first magnet member is eliminated, thereby loosening the clamping of the second port and the filter membrane, allowing them to move to the second position under the action of gravity.

[0127] In a specific embodiment, the cell culture bag further comprises an inlet port and an exhaust port, wherein the inlet port is used to be in fluid communication with an external gas source. In a preferred embodiment, the exhaust port is in fluid communication with a pressure control valve such as a one-way valve located outside the cell culture bag, and an air filter is further provided between the exhaust port and the pressure control valve, and a heater is provided on the outside of the air filter for heating the gas discharged from the exhaust port.

[0128] In some specific embodiments, the rapid flow bioreactor further comprises a temperature sensor, a pH sensor and a DO sensor inserted into the cell culture bag, wherein preferably, a flexible sleeve is provided on the outside of the temperature sensor so that the temperature sensor is not in direct contact with the culture medium. In some preferred embodiments, the rapid flow bioreactor further comprises a temperature regulating module, a pH regulating module, a DO regulating module and a gas flow regulating module. In some more preferred embodiments, the rapid flow bioreactor further comprises a control unit, which receives signals from corresponding sensors and issues notifications and / or operates corresponding regulating modules to adjust corresponding indicators according to preset programs.

[0129] In other aspects, in addition to including sensors / probes and one or more connections (such as tubes, valve holes) for fluids and gases, etc. to enter the channel of the cell culture bag, the torrent bioreactor of the present invention may also include one or more pumps, controllers and electronic devices (such as heating devices, cooling devices, acid reagent sources, alkaline reagent sources, defoaming agent sources, pressurization / pressure relief devices, sensor electronic devices, pressurized gas controllers, liquid (such as culture medium) sources, etc.). Important operating index parameters monitored and controlled by sensors / probes and controllers may include any one or more combinations of temperature, movement mode and speed, pH, DO, airflow rate (for example), etc. Those skilled in the art are capable of making routine adjustments to known components and control strategies within the scope of known torrent bioreactors according to their functions. In a preferred embodiment, the iPSC culture method of the present invention is carried out in a torrent bioreactor of the present invention.

[0130] Quality Control Standards for Bioreactor Culture of Induced Pluripotent Stem Cell Aggregates

[0131] The most intuitive standard for qualified reactor culture of induced pluripotent stem cell aggregates is the growth picture of the aggregates. The cells will form balls within the first day after inoculation, but the early morphology is very irregular. As time goes by, it will regularly grow larger and larger, and the edges will gradually become regular. Whether in the early or later culture process, shear force damage has a great influence on the maintenance of aggregate morphology and state. In order to reduce the shear force on the aggregates, it is necessary to analyze the shear force distribution generated by reactor vibration or stirring. In general, we need to allow single cells to self-assemble into small balls with tight edges, roundness, and uniform size, and then perform subsequent differentiation and related stability studies after reaching a proliferation multiple equivalent to or even better than the wall-attached process. In addition, we can also compare the reactor-cultured induced pluripotent stem cell aggregates with positive samples of the wall-attached or suspended manual maturation process by methods such as qPCR, FACS, ICC, and single-cell sequencing.

[0132] In some embodiments, the iPSC culture method of the present invention further comprises a quality control / quality inspection step for the harvested cells. Preferably, the quality control / quality inspection results show that the pluripotency of the cells is substantially consistent with that of manual 2D culture.

[0133] Uses of cultured iPSCs

[0134] In some embodiments, the iPSC culture method of the present invention further includes using the harvested cells for further inoculation, freezing, differentiation, clinical application or other scientific research purposes. In some specific embodiments, the iPSC culture method of the present invention further includes using the harvested cells for differentiation to produce iPSC-derived cells or iPSC-derived cell colonies. In some specific embodiments, the iPSC culture method of the present invention further includes using the harvested cells for differentiation to produce iPSC-derived cells or iPSC-derived cell colonies, and preparing iPSC-derived cell products.

[0135] In the art, the term "iPSC-derived cells" refers to other iPS cells or differentiated cells produced from iPSC by in vitro amplification and / or differentiation procedures, for example, immune cells (T cells, NK cells or macrophages), cardiomyocytes, hepatocytes, neural cells, and their precursor cells. The type of the cell can be determined by detecting the gene expression profile or marker gene expression of the differentiated cells. As used herein, the term "differentiation" refers to a method in which iPSC is subjected to a cell progressing from an undifferentiated state to a differentiated state. Typically, the step of differentiating iPSC includes culturing cells under specific conditions and / or contacting the cells with certain factors.

[0136] In the art, the term "iPSC-derived cell colony" refers to a cell colony comprising the differentiated iPSC-derived cells derived from iPSC. The cell colony may be a cell colony formed by differentiation from iPSC using an in vitro differentiation procedure, such as an iPSC-derived cell sample. The cell colony may also be a cell colony composed of iPSC-derived cells determined based on artificial annotations. Therefore, in some aspects, the iPSC-derived cell colony may be a sample-based iPSC-derived cell colony. In other aspects, the iPSC-derived cell colony may be an annotation-based iPSC-derived cell colony.

[0137] In the art, the term "iPSC-derived cell product" refers to a product comprising an iPSC-derived cell population. Such a cell-derived product can be transferred to a source mammalian individual of the somatic cells used to produce the iPSC. In other words, the somatic cells used in the method of the present invention can be autologous cells. Alternatively, the somatic cells used in the method of the present invention can be allogeneic relative to the mammalian individual to which the downstream cell-derived product is administered. Preferably, the somatic cells used in the method of the present invention are derived from patients to be treated with cell-derived products, i.e., autologous cells. In some embodiments, the product is a product comprising an iPSC-derived cell population, or substantially consisting of the cell population, consisting thereof, especially a pharmaceutical product, such as a regenerative pharmaceutical product. In some embodiments, the product is suspected or known to contain a residual amount of undifferentiated iPSC. It is understandable that, based on the intended application purpose of the product, the product can be in any suitable form. For example, the product can be a composition comprising an iPSC-derived cell population and optionally a pharmaceutically acceptable excipient.

[0138] The somatic cells, as well as the corresponding induced iPSCs and derived downstream cells, can be autologous cells or allogeneic cells. Example

[0139] Experimental Materials

[0140] Table 1 Key instrument and equipment information

[0141]

[0142] Table 2 Key reagents and consumables information

[0143]

[0144]

[0145] Example 1 Torrent Bioreactor

[0146] The torrent bioreactor rotates horizontally around an axis at a set rate, so that the liquid culture medium is agitated and a thin film with high gas-liquid mass transfer efficiency is formed on the side wall of the cell culture bag. The film is then saturated with oxygen in the top space and combined with the liquid body. This process is repeated over and over again to provide a strong oxygen mass transfer efficiency. In addition, since the torrent bioreactor does not contain stirring and foaming devices, the shear force on cells is very low. In addition, the gas-liquid mass transfer efficiency is high, the parameter control is precise, the experimental operation is simple, and it also has good scalability. It can cleverly avoid the shortcomings of wave reactors and stirring reactors, and is an ideal reactor type for iPSC culture.

[0147] In addition, in order to better culture iPSC cells, the present invention performs one or more of the following customization and optimization strategies based on the existing torrent bioreactor in the art:

[0148] (1) For the fluid replacement during the aggregate culture process, a dedicated fluid replacement pipeline is added, which is suitable for efficient fluid replacement from the formation of aggregates to their growth to a larger size;

[0149] (2) filling the bag with gas and raising the air pressure appropriately, thereby ensuring that the bag is inflated rather than relaxed, so as to improve the homogeneity of the movement pattern of the aggregate;

[0150] (3) The temperature sensor (electrode) is inserted into the bag or the flexible sleeve, so that the temperature of the culture medium in the rapid flow can be continuously monitored online without direct contact with the culture medium to avoid contamination risks;

[0151] (4) A more gentle means of pH adjustment, achieved by introducing different gases into the culture medium instead of acidic / alkaline reagents;

[0152] (5) A control unit that can independently control DO, pH value, temperature, rotation speed, air intake, exhaust gas, liquid inlet, liquid outlet, and liquid exchange, and can set different combined control strategies according to different culture stages.

[0153] Figure 1The structural schematic diagram of an example of a torrent bioreactor 10 according to the present invention is shown. As can be seen from the figure, in this embodiment, the torrent bioreactor 10 includes a reactor tank 1, a cell culture bag 2 and a driver 3. In order to clearly show the specific structure of the cell culture bag 2, the reactor tank 1 in the figure is partially cut, and the cell culture bag 2 is shown in a transparent form. The reactor tank 1 has a cylindrical body and a conical bottom. The size and volume of the cell culture bag 2 are equivalent to the inside of the reactor tank 1. When in use, it is installed in the reactor tank 1 and fixed, for example, by a plurality of clamps 25 on the top surface. It includes a flexible bag body 20 for accommodating cell culture, a liquid inlet pipeline 21 for injecting culture medium, a liquid outlet pipeline 22 for removing culture medium, a dedicated liquid exchange pipeline 23 and a holding mechanism 24 for the dedicated liquid exchange pipeline. The driver 3 can be arranged, for example, below the reactor tank 1 (for example, in the base), for driving the reactor tank 1 to perform horizontal circular motion around a central axis, so that the liquid culture medium in the cell culture bag generates torrents in this operating mode. Here, it should be understood that in different culture stages, the driver 3 can use different rotation rates to drive the operation of the reactor tank 1. For example, when balancing the culture medium, the rotation rate can be 40-50rpm; when inoculating single cells, the rotation rate can be 40-50rpm; after the aggregate is replaced with liquid, the rotation rate can be 45-55rpm. These operating indicators can be arbitrarily adjusted by the operator within the range of hardware support according to needs or experimental design.

[0154] In the above example according to the present invention, the dedicated liquid exchange pipeline 23 includes a first port 231 extending outside the flexible bag body, a second port 232 extending inside the flexible bag body, and a filter membrane 233 attached to the second port, and the holding mechanism 24 is advantageously configured to enable the second port 231 and the filter membrane 233 to move from a first position to a second position, wherein in the first position, the second port and the filter membrane are held to be located above the liquid level L of the culture medium without contacting the culture medium; in the second position, the second port 231 and the filter membrane 233 are located below the liquid level L of the culture medium. Compared with the torrent bioreactor in the prior art, the provision of the dedicated liquid exchange pipeline 23 is particularly advantageous for the culture of iPSC aggregates, and the dedicated liquid exchange pipeline 23 is used for liquid exchange when the aggregates in the cell culture bag 2 are small in size, for example, 100 μm to 150 μm, and difficult to settle quickly (for example, within about 5 minutes). In this case, both the inlet and outlet are carried out using the liquid exchange pipeline 23. The aggregates will be retained on the filter membrane 233 when the old culture medium is discharged, and will be flushed back into the culture system with the new culture medium. Therefore, there will be no influence of dead cells attached to the wall of this liquid exchange pipeline.

[0155] See again Figure 1 and Figure 2In a preferred embodiment of the holding mechanism 24, the holding mechanism 24 includes a first magnet 241 located inside the flexible bag, a second magnet 242 located outside the flexible bag, and a fixing member 243 for the first magnet. In the first position, the first magnet and the second magnet clamp the second port and the filter membrane to the top side wall of the flexible bag through magnetic force. Before the dedicated liquid exchange pipeline 23 is used to perform the liquid exchange operation, the second magnet 242 is removed to eliminate the magnetic force on the first magnet, thereby loosening the clamping of the second port and the filter membrane, and allowing them to move to the second position under the action of gravity. As an example, in this example, the first magnet and the second magnet are both magnets. Since the second magnet is located on the outside of the flexible bag, any operation on it will not affect the sterile state inside the bag. For example, when the iPSC single cells in the cell culture bag are aggregated and in good condition (with tight edges and regular morphology), the second magnet is removed. At this time, the first magnet located on the inside of the flexible bag naturally falls off into the culture system due to gravity, and then the end of the dedicated liquid exchange pipeline with the filter membrane also drops below the liquid level L of the culture medium due to the weight, so that the liquid exchange pipeline switches to a usable working state.

[0156] exist Figure 1-2 In the example shown in , the fixing member 243 can be a tether, one end of which is connected to the first magnet member 241, and the other end is connected to the temperature sensor 9 inserted into the cell culture bag 2 and fixed (it can also be appropriately fixed to other positions). A flexible sleeve 90 is provided on the outside of the temperature sensor 9, so that the temperature sensor does not directly contact the culture medium, reducing the risk of contamination.

[0157] In the present invention, in order to ensure that the liquid can be replaced at a ratio of 50% to 75% and that the movement pattern of the aggregates will not be affected, the second port and the second position of the filter membrane are preferably at the lower quarter to one-third height of the reactor tank body 1, that is, from the bottom of the reactor tank body to the lower quarter to one-third height of the tank body, and the surface area of ​​the filter membrane is also fixed, and the pore size of the filter membrane 233 can be 20μm to 100μm, preferably 20μm to 60μm. In the later stage of culture, when the size of the aggregates is larger, for example, greater than 150μm, preferably greater than 300μm, the liquid replacement efficiency of the dedicated liquid replacement pipeline 23 will be significantly reduced, for example, the filter membrane will be more easily blocked. Therefore, the liquid inlet pipeline 21 and the liquid outlet pipeline 22 will be more preferred means of liquid replacement.

[0158] In the torrent bioreactor 10 according to the present invention, the cell culture bag 2 also includes an air inlet port 4 and an exhaust port 5. The air inlet port 4 can be used to communicate with an external gas source (not shown) fluid, and the exhaust port 5 can be connected to a pressure control valve 6 such as a one-way valve located outside the cell culture bag 2, and an air filter 7 is also provided between the exhaust port and the pressure control valve. A heater 8 is provided on the outside of the air filter to heat the gas discharged from the exhaust port 5 to avoid the condensed water generated by the tail gas from flowing back, causing pollution and safety accidents. The pressure control valve 6 can keep the air pressure in the bag body in an appropriately elevated state, so that the flexible bag body remains bulging and has no wrinkles, and the pressure control range of the pressure control valve 6 includes at least 500Pa to 2000Pa. In addition, the downstream end of the pressure control valve can also be connected to the tail gas bottle b. In addition, an air filter can also be connected upstream of the air inlet port 4, and the gas in the external gas source can enter the cell culture bag 2 at a specific ratio and speed through the controller and the air filter.

[0159] In this example, see Figure 3 , the torrent bioreactor 10 may also include a pH sensor 11, a DO sensor 12, a sampling port 14 and spare pipelines 13, 15. The torrent bioreactor also includes a temperature regulating module, a pH regulating module, a DO regulating module, and a gas flow regulating module which are not marked in the figure, and more preferably also includes a control unit, which receives signals from corresponding sensors and operates corresponding regulating modules according to preset programs to adjust corresponding indicators. The above components are conventional torrent sensor components known in the art, and their structures have been fully described in the prior art (although they may be different from this example), and those skilled in the art know how to structurally set these reactor components to achieve their basic functions.

[0160] It should be understood that the positions of the components shown in the figure are merely for illustration purposes and are not necessarily intended to limit the scope of the present invention. Figure 1 The positions of the components shown in the figure correspond to each other. Those skilled in the art are capable of making routine adjustments to the known components within the scope of the known turbulent flow bioreactor according to their functions.

[0161] Example 2 Stirred Bioreactor

[0162] In order to achieve better culture effect of iPSC in a stirred bioreactor and realize the operation mode of making the culture medium move in horizontal circular motion during the culture process, the inventors customized and optimized the stirring blades based on the existing conventional stirred bioreactor.

[0163] Figure 4A schematic structural diagram of an example of a stirred bioreactor 10' of the present invention is shown. As can be seen from the figure, like the stirred bioreactors already known in the art, the stirred bioreactor of the present invention includes a cylindrical tank body 1', an agitator 2' and a drive motor 3' for driving the agitator to rotate. The cylindrical tank body 1' defines a cylindrical chamber 22' for accommodating cell culture, and a cover 11' is provided on the top of the cylindrical chamber. In this embodiment, the agitator 2' has a stirring shaft 20', the stirring shaft having a first end located outside the cylindrical chamber and a second end where the cover extends into the cylindrical chamber, wherein the first end is connected to the drive motor 3', and a stirring paddle 21' is fixedly mounted on the shaft, substantially located at the second end. Driven by the drive motor 3', the rotation speed of the stirring paddle can be, for example, 80-140rpm.

[0164] Figure 5 The schematic structural diagram of another embodiment of the stirred bioreactor 10' of the present invention is shown, wherein two stirring paddles 211' and 212' are fixedly mounted on the stirring shaft, wherein 212' is substantially located at the second end, and 211' is located at a position slightly higher than 212' on the shaft, and the two stirring paddles have the same shape and size, and can also be independently set to different sizes. In addition, more stirring paddles can also be installed.

[0165] See also Figure 7 A and Figure 7 B shows a preferred embodiment of the customized optimized stirring paddle 21' of the present invention. The stirring paddle 21' comprises a cylindrical sleeve 24' having a central axis and three stirring blades 23' evenly distributed along the periphery of the cylindrical sleeve, wherein each of the stirring blades has an angle of 45° with a plane perpendicular to the central axis, and the ratio of the diameter of the stirring blade to the diameter of the cylindrical chamber is 0.3 to 0.9, preferably 0.4 to 0.8. For example, the diameter of the stirring blade of this example is selected to be 4 cm; the diameter of the cylindrical chamber is 8 cm, and the volume can be, for example, 300 mL to 500 mL or even larger to accommodate a suitable culture volume.

[0166] In this example, see Figure 6, the stirred bioreactor 10' also includes an inlet pipeline 12' and an outlet pipeline 13' extending into the cylindrical chamber 22' through the cover 11'; and air inlet ports 141' and 142' (bottom-through air inlet and surface-through air inlet, respectively), an exhaust port 15' and a sampling port 16' arranged in the cover and connected to the cylindrical chamber fluid. The inlet pipeline is used to inject the culture medium into the cylindrical chamber, and the outlet pipeline is used to remove the culture medium or harvest cells; the inlet port 14' is connected to the external air filter, and the gas from the external gas source enters the cylindrical chamber at a specific ratio and speed through the controller and the air filter; the downstream of the exhaust port 15' can be connected to a tail gas bottle (not shown). And the stirred bioreactor 10' also includes a temperature sensor 17', a pH sensor 18' and a DO sensor 19' inserted into the cylindrical chamber. Advantageously, a sleeve is provided outside the temperature sensor 17' so that the temperature sensor does not come into direct contact with the culture medium to avoid contamination, and the sleeve is a stainless steel sleeve or a flexible sleeve, preferably a stainless steel sleeve. Preferably, the stirred bioreactor further includes a temperature adjustment module, a pH adjustment module, and a DO adjustment module, and more preferably also includes a control unit, which receives signals from corresponding sensors and operates corresponding adjustment modules to adjust corresponding indicators according to a preset program. In this example, a foam sensor 26' is also included.

[0167] In the above-mentioned stirred bioreactor 10' according to the present invention, due to the use of optimized stirring blades, it can generate a liquid flow mainly in the circumferential direction in the culture medium, and mix a certain radial flow and axial flow. This combined flow can achieve a better mixing effect and the shear force generated is also small, which is suitable for the suspended growth of iPSC aggregates.

[0168] When performing a liquid replacement operation in the stirred bioreactor 10' of the present invention, a cell filter bag externally connected to the cylindrical chamber 22' is used. The cell filter bag is also a conventional device already available in the art.

[0169] It should be understood that the positions of the components depicted in the figure only illustrate the situation of this example, and their distribution is exemplary. Those skilled in the art are capable of making routine adjustments to the known components within the scope of known stirred bioreactors according to their functions. Example 3 Suspension culture of iPSCs using a torrent bioreactor

[0170] 3.1 Process flow

[0171] Prepare the torrent bioreactor as described in Example 1, and put it into the matching disposable culture bag, and connect the various pipelines and electrodes according to the settings.

[0172] One day before inoculating cells, place the culture medium into the bioreactor with a culture volume of 600 mL, using hPSC-AOF medium throughout the process. Then pre-equilibrate the medium according to the culture conditions below.

[0173] Human induced pluripotent stem cells (iPSCs) were seeded / passaged into culture medium at a density of 1.5E5 viable cells / mL (not exceeding 3.5E5), with the day of seeding / passaging being Day 0. After seeding / passaging, 10 μM Rocki was added to the culture medium.

[0174] Samples were taken every day from Day 1 to Day 4, and the cell medium was replaced by about 50% of the volume (ie, half-volume medium replacement).

[0175] The culture conditions were controlled and maintained as follows: During the culture and sampling process, the reactor speed was monitored and maintained at 40-80 rpm, and the temperature of the culture system was monitored and maintained at about 37°C. The pressure in the bag was controlled to about 500 Pa by the pressure control valve at the tail gas end, so that the bag was kept inflated and continuously heated by the tail gas heater. The DO value of the culture system was maintained at 5-80%, and the pH was maintained between 6.8 and 7.4. For this reason, air was passed through at a flow rate of 30 mL / min-80 mL / min during pre-equilibrium, 50 mL / min-100 mL / min during inoculation, and 100 mL / min-300 mL / min during passage. Because iPSC aggregates require milder culture conditions, acidic and alkaline reagents were avoided as much as possible throughout the process. Air was passed through when the pH value decreased or was too low, and CO2 gas was passed through when it increased or was too high.

[0176] Within about 24 hours after the cells are seeded into the culture medium, iPSCs gradually grow and proliferate from mainly single cells to smaller aggregates, for example, aggregates smaller than about 100 μm. In this state, the cells gradually accumulate growth-promoting substances during the growth process, so it is preferred not to perform any medium replacement operation, which is more conducive to promoting cell growth.

[0177] Starting earlier or later on Day 1, most cells will gradually grow and proliferate in the culture medium into aggregates with a diameter of more than 100 μm, and more between 100 μm and 150 μm. Therefore, starting from Day 1, a cell exchange ratio of about 50% volume is performed through a specially added liquid exchange pipeline, wherein the filter retains the cell aggregates to prevent them from being removed from the culture system with the waste liquid, and is flushed back into the culture system by the new culture medium added later. The amount of liquid exchange is 25% to 75% of the volume of the culture system, preferably about 50%. Preferably, the liquid exchange does not change or substantially change the volume of the culture system. During the liquid exchange through the dedicated liquid exchange pipeline, in order to avoid aggregate fusion, the speed cannot be reduced or even stopped, and it is preferably appropriately increased. After the liquid exchange is completed, the operating speed of the reactor is kept at least about +5 rpm faster, and after running for at least 5 minutes, it is gradually adjusted back to the original speed.

[0178] In the late stage of culture, when the cells exist in the culture medium as aggregates with a diameter greater than 150 μm, they can settle in a short time. Therefore, the liquid replacement operation is preferably changed to a conventionally set inlet / outlet dual pipeline as a liquid replacement channel, and a cell liquid replacement of about 50% volume ratio is performed, wherein at least about 5 minutes before the start of the liquid replacement, the bioreactor movement is stopped to allow the aggregates to settle in the culture medium to the lower half or even the bottom of the culture system, and the liquid replacement is completed by extracting the old culture medium in the upper half of the culture system and adding new culture medium. The liquid replacement volume is 25% to 75% of the volume of the culture system, preferably about 50%, and preferably the liquid replacement does not change or substantially change the volume of the culture system. Alternatively, the liquid replacement operation can still be performed through a dedicated liquid replacement pipeline, although its liquid replacement efficiency is reduced.

[0179] When the culture reaches Day 4, all the culture fluid, i.e., the aggregate suspension, is harvested for the next passaging or cryopreservation. The procedure is as follows: all the aggregate suspension is collected into a receiving bottle connected to the cell culture bag, and a certain volume of the aggregate suspension is transferred to a centrifuge tube for dissociation. Before transferring the suspension, the receiving bottle maintains horizontal circular motion for about 1 to 3 minutes to evenly suspend all the aggregates in the bottle; the supernatant is discarded, DPBS is added, and the supernatant is discarded after sedimentation for 1 to 10 minutes, and a 2 to 4-fold diluted dissociation solution is added. The dissociation is performed for 5 to 20 minutes. After the aggregates are dissociated into a homogeneous single-cell suspension, the culture medium is added to terminate the reaction, and the cells are harvested; the harvested cells are used for further passaging, differentiation or cryopreservation.

[0180] In addition, a shake flask control group was set up for the above-mentioned whole process: the inoculation density, culture cycle, liquid change ratio, culture medium and reactor experimental group were consistent, the culture container was a 125mL conical shake flask, the culture volume was 15-40mL, and the dissociation solution composition, dissociation procedure and reactor experimental group were consistent.

[0181] 3.2 Experimental Results

[0182] The rapid flow bioreactor culture process of iPSC aggregates can be equivalent to the artificial culture process. The details are as follows.

[0183] 3.2.1 Cell growth results

[0184] Take pictures with an inverted biological microscope to observe and analyze cell growth pictures ( Figure 8 ), the microscope B used on Day 1, Day 2, and Day 3 was Carl Zeiss Primovert (scale unit: 5 mil), and the microscope A used on Day 4 was Nexcope NIB610 (scale unit: 100 μm), with magnifications of 10×10. Figure 8 It shows that after the cells are inoculated in the torrent bioreactor (Day 0), the particle size on Day 1 is slightly smaller than that of the aggregates cultured in shake flasks, but the aggregates proliferate faster. The culture continues to Day 2 and Day 3, and the particle size is getting closer and closer to that of the aggregates cultured in shake flasks. On the day of harvest, the particle size of the aggregates cultured in the reactor is basically the same as that of the aggregates cultured in shake flasks. In order to reduce the impact of the free liquid surface on the aggregates, the aggregates are distributed at the middle and lower liquid levels during the reactor culture process. The edge compactness, morphological roundness and particle size uniformity of the aggregates on the day of harvest are basically the same as those of the aggregates cultured in shake flasks. In addition, the growth pictures of induced pluripotent stem cell aggregates cultured in two consecutive batches of reactors are basically the same, and it can be considered that the morphological indicators of induced pluripotent stem cell aggregates cultured in reactors are relatively stable.

[0185] 3.2.2 Particle size analysis

[0186] The cell growth pictures of Example 3.2.1 were sorted, and the diameter of the aggregates was detected by image J to analyze the uniformity of the particle size. These curves are the aggregate diameter data of each cell picture detected, and the percentage of the number of aggregates in different particle size ranges to the total number of aggregates is calculated. The results are shown in Figure 2. Fig. 9 As shown in the figure, the aggregates cultured in the torrent bioreactor and shake flask showed a normal distribution in the 4-day culture period and were concentrated in the middle range, with the aggregates in the reactor and shake flask culture ranging from 80 to 200 μm and 180 to 240 μm, respectively. In addition, the columnar analysis of the data showed that the aggregates cultured in the reactor were slightly smaller in diameter in the early stage, and the aggregates in the reactor grew slightly faster during the culture to Day 4 until their particle size was basically the same as that of the aggregates cultured in the shake flask. In addition, the particle size index of the induced pluripotent stem cell aggregates cultured in two consecutive batches of reactors was relatively stable.

[0187] 3.2.3 Analysis of doubling times

[0188] The formula for calculating the multiplication factor is: Fold = Harvest-VCD / Seed-VCD, where Harvest-VCD is the viable cell concentration of the aggregates cultured in the reactor when the cells are harvested, and Seed-VCD is the cell seeding density, that is, 1.5E5 viable cells / mL. The results are shown in Table 3, that is, the cell doublings in the reactor and 125mL shake flask are basically the same. In addition, the proliferation multiples of induced pluripotent aggregates cultured in 500mL shake flasks are significantly lower than those in 125mL shake flasks and reactor culture systems. Considering that multi-container culture will increase the heterogeneity and cell loss of cells within the batch during large-volume culture, and the multiplication multiples of the reactor experimental group are equivalent to those of the 125mL shake flask, therefore, the torrent bioreactor suspension culture is the preferred choice for large-scale culture of induced pluripotent aggregates.

[0189] Table 3 Cell proliferation analysis

[0190]

[0191] *VCD: viable cell density

[0192] 3.2.4 qPCR results analysis

[0193] The pluripotency of iPSCs obtained by culture in Example 3 was detected by qPCR. Specific method: RNA was extracted using a total RNA extraction kit (OMEGA), and then TM RT Master Mix (Takara) was used to reverse transcribe into cDNA, and finally TB Green TM Premix Ex Taq TM Ⅱ (Takara) was used for qPCR detection in a fluorescence quantitative PCR instrument (Roche, Z480), and the pluripotency markers detected were OCT4, NANOG and TRA-1-81. Fig.10 As shown, the pluripotency indicators of iPSC aggregates cultured in torrent bioreactor and shake flask were basically consistent.

[0194] 3.2.5 FACS analysis

[0195] The pluripotency of iPSCs obtained by culture in Example 3 was detected by FACS method. Specific method: The cells were collected in a centrifuge tube and centrifuged. Accutase was used to digest the cells at 37°C for 5 min. The digested cells were resuspended in FACS buffer, the cells were counted and the survival status was determined. The cells were stained with fluorophore-conjugated monoclonal antibodies: anti-human NANOG, anti-human TRA-1-60, anti-human TRA-1-81, anti-human OCT4, anti-human SSEA-4 and anti-human SOX2. The analysis of living cells was performed on a flow cytometer (Beckman Coulter).

[0196] The results are as follows Fig.11 As shown, the pluripotency indexes of iPSC aggregates cultured in the rapid flow bioreactor were better (>90%).

[0197] Example 4 Suspension culture of iPSCs using a stirred bioreactor with optimized blades

[0198] 4.1 Process flow

[0199] This example uses a stirred bioreactor, the reactor is as shown in Example 2 and Figure 4-7 The operation steps and culture conditions are as shown in Example 3.1, except for the following:

[0200] hPSC-Scale medium was used throughout the process, and the culture volume was 300 mL. During the culture and sampling process, the stirring blade speed was monitored and maintained at 80-140 rpm, DO was 5-80%, and pH was 6.8-7.4.

[0201] First, the operation test was carried out: the DO, pH and temperature regulation of the equipment itself were relatively stable; the temperature could be stabilized within 30 minutes, and the DO and pH could be stabilized within 30 to 60 minutes. The ventilation volume and the ventilation ratio of the four-way gas required to maintain the culture conditions of DO 5 to 80% and pH 6.8 to 7.4 were preliminarily determined. The pre-culture was carried out at a stirring paddle speed of 80 to 140 rpm, and the growth state was good, with no obvious damage on the edge.

[0202] The fluid exchange scheme also refers to the torrent bioreactor. When the aggregate size is relatively small, since it is not easy to directly set up a dedicated fluid exchange pipeline in the tank body, the fluid is changed through an external additional cell filter bag. Before the fluid exchange, the operation of the reactor is suspended, and the culture medium is extracted into the external cell filter bag. The aggregates are retained by filtration and the used culture medium is removed. Then, a roughly equal volume of fresh culture medium is added, and finally the culture that has completed the fluid exchange is returned to the tank body. When the aggregate size is large, the fluid can be exchanged by sedimentation, which is performed through the inlet and outlet pipelines in a similar manner to the operation in Example 3.1.

[0203] The timing and operation of cell harvesting were also carried out according to the protocol in Example 3.1 in a torrent bioreactor.

[0204] 4.2 Experimental Results

[0205] The iPSC aggregate culture process using a stirred bioreactor with optimized paddles can effectively replace the artificial culture process.

[0206] 4.2.1 Cell growth results

[0207] An inverted microscope was used to take pictures and observe and analyze the cell growth images. The control group used microscope B and the reactor used microscope A, and the magnification was 10×10. Fig.12 As shown in the figure, after the cells of the stirred bioreactor with optimized blades were inoculated (Day 0), the particle size was significantly smaller than that of the aggregates in the shake flask culture (the control group in the figure), but the aggregates proliferated faster and continued to be cultured until the day of harvest on Day 4, which was significantly larger than the aggregates in the shake flask culture. The edge compactness, morphology and roundness of the aggregates cultured in the reactor were basically the same as those of the aggregates cultured in the shake flask on the day of harvest. In addition, it can be seen with the naked eye that the aggregates are evenly distributed inside the tank during the culture process.

[0208] 4.2.2 Particle size analysis

[0209] The cell growth pictures of Example 4.2.1 were collated, and the diameter of the aggregates was detected by image J to analyze the uniformity of the particle size. These curves are the aggregate diameter data of each cell picture detected, and the percentage of the number of aggregates in different particle size ranges to the total number of aggregates is calculated. The results are shown in Figure 2. Fig.13 As shown in the figure, the aggregates of the stirred bioreactor with optimized blades and the shake flask culture (marked as ctl in the figure) showed a normal distribution in the 4-day culture period and were concentrated in the middle range of particle size, where the aggregate size ranges of the reactor and shake flask culture were about 70-320μm and 160-250μm, respectively. In addition, the columnar analysis of the data showed that the diameter of the aggregates in the reactor culture in the early stage (Day1-Day3) was always smaller, and until Day4, the aggregate size of the reactor culture was significantly higher than that of the shake flask culture.

[0210] 4.2.3 Analysis of doubling times

[0211] The formula for calculating the multiplication factor is: Fold = Harvest-VCD / Seed-VCD, where Harvest-VCD is the viable cell concentration of the aggregates cultured in the reactor when the cells are harvested, and Seed-VCD is the cell seeding density, that is, 1.5E5 viable cells / mL. The results are shown in Table 4, that is, the cell doublings in the reactor and 125mL shake flask are basically the same. In addition, the proliferation multiples of induced pluripotent aggregates cultured in 500mL shake flasks are significantly lower than those in the 125mL shake flask and reactor culture systems. Considering that multi-container culture will increase the heterogeneity and cell loss of cells within the batch during large-scale culture, and the doubling multiples of the reactor experimental group are the largest, therefore, this stirred bioreactor suspension culture with optimized blades is the preferred choice for large-scale culture of induced pluripotent aggregates.

[0212] Table 4 Cell proliferation analysis

[0213]

[0214] 4.2.4 FACS analysis

[0215] The pluripotency of the iPSCs obtained by culture in Example 4 was detected by FACS method. The method was the same as that described in 3.2.5. Fig.14 The results showed that the pluripotency indicators of iPSC aggregates cultured in the stirred bioreactor with optimized blades were better (>90%).

[0216] Example 5 Comparative Example

[0217] Comparative Example 1 Horizontal stirring paddle bioreactor

[0218] 5.1.1 Process flow

[0219] Reactor Fig.21 As shown, the paddle is a horizontal two-blade (with baffle) paddle, the process flow is consistent with that of Example 4.1, and the culture medium used throughout the process is hPSC-AOF.

[0220] 5.1.2 Experimental Results

[0221] 5.1.2.1 Cell growth results

[0222] Inverted microscope A was used to take pictures, and the cell growth pictures were observed and analyzed, with the magnification being 10×10. Fig.15As shown in the figure, after the cells were inoculated (Day 0) in the horizontal stirring paddle bioreactor, the particle size on Day 1 was significantly smaller than that of the aggregates in the shake flask culture and the proliferation was very slow. A small amount of aggregate fusion was produced on Day 2, and floccules were obviously produced on Day 3. The growth state was poor and the culture was terminated on Day 4. During the culture process, it was found that the speed was adjusted to the limit within the adjustable and low shear speed range, and all the aggregates were concentrated in the central area of ​​the lower middle part of the tank, that is, the mixing effect was extremely poor. It is suggested that the horizontal stirring reactor does have high requirements for the blades, and a good mixing effect is required to achieve the suspension culture of iPSC aggregates. This type of horizontal stirring paddle bioreactor is not suitable for the culture of induced pluripotent aggregates.

[0223] 5.1.2.2qPCR detection and analysis

[0224] The pluripotency of iPSCs cultured by the method of Comparative Example 1 was detected by qPCR. The method was the same as that disclosed in 3.2.4. The results are shown in Fig.16 As shown, the pluripotency indicators of iPSC aggregates cultured in horizontal agitator bioreactors and shake flasks were poorer, indicating that the culture process affected the characteristics of the cells.

[0225] Comparative Example 2 Wave Bioreactor

[0226] 5.2.1 Process flow

[0227] Reactor Fig. 22 As shown, the process flow is basically the same as that of Example 3.1, but the culture days are increased to Day 5 (Day 1-5), and the culture medium used throughout the process is hPSC-AOF.

[0228] 5.2.2 Experimental Results

[0229] 5.2.2.1 Cell growth results

[0230] Inverted microscope B was used to take pictures, and the cell growth pictures were observed and analyzed, with the magnification being 10×10. Fig.17 As shown in the figure, after the cells were inoculated in the wave bioreactor (Day 0), the particle size on Day 1 was significantly smaller than that of the aggregates in the shake flask culture and the proliferation was slower. On Day 2, aggregate fusion and attachment to the culture bag wall and rapid accumulation began to occur, and the culture was terminated on Day 5. During the culture process, it was found that the aggregates moved in the lower central area of ​​the culture bag and there was no liquid surface raised at both ends of the culture bag. The cell growth morphology was ellipsoidal with poor uniformity.

[0231] 5.2.2.2 Analysis of doubling times

[0232] The formula for calculating the multiplication factor is: Fold = Harvest-VCD / Seed-VCD, where Harvest-VCD is the viable cell concentration of the aggregates cultured in the reactor at the time of cell harvest, and Seed-VCD is the cell seeding density, i.e., 1.5E5 viable cells / mL. The results are shown in Table 5, that is, the multiplication of the aggregates cultured in the reactor is significantly lower than that of the aggregates cultured in 125mL and 500mL shake flasks, that is, this type of motion reactor process is not a good method for aggregate amplification.

[0233] Table 5 Cell proliferation analysis

[0234]

[0235] 5.2.2.3qPCR detection and analysis

[0236] The pluripotency of iPSCs cultured by the method of Comparative Example 2 was detected by qPCR. The detection method was the same as that disclosed in 3.2.4. The results are shown in Fig.18 As shown, the pluripotency indicators of iPSC aggregates cultured in wave bioreactors were inferior to those in shake flasks.

[0237] Example 6 Application of iNK cell differentiation

[0238] The iPSC cells obtained in Example 4 were used to continue to be cultured for NK cell differentiation in the reactor.

[0239] The specific steps of flow cytometry detection are as follows: Collect cells in a centrifuge tube and centrifuge. Use Accutase to digest cells at 37°C for 5 minutes. Resuspend the digested cells in FACS buffer, count the cells and determine the survival status. Stain the cells with a fluorophore-conjugated monoclonal antibody: anti-human CD34 antibody. Perform the analysis of live cells on a flow cytometer (Beckman Coulter).

[0240] The results are as follows: iPSCs differentiated normally into hematopoietic stem cells (HPCs): The flow cytometry results on Day 16 showed that CD34 + The proportion of positive cells was 89.56% ( Fig. 20 ), and the free single cell concentration reached a maximum value on Day 22: 3.47E6 cells / mL ( Fig.19 ), growth and differentiation are in good condition.

[0241] Therefore, the reactor and culture method of the present invention can produce iPSC aggregates and be used for differentiation of iNK cells or other cells.

Claims

1. A torrent bioreactor, characterized in that: The turbulent bioreactor (10) comprises: Reactor tank (1); a cell culture bag (2) fixedly installed in the reactor tank (1), the cell culture bag comprising a flexible bag body (20) for accommodating cell culture, a liquid inlet pipeline (21) for injecting culture medium, a liquid outlet pipeline (22) for removing culture medium, a liquid exchange pipeline (23) and a holding mechanism (24) for the liquid exchange pipeline; and A drive (3) connected to the reactor vessel, the drive being configured to drive the reactor vessel (1) to rotate horizontally, thereby generating a turbulent flow in the cell culture bag; The liquid exchange pipeline (23) includes a first port (231) extending outside the flexible bag body, a second port (232) extending outside the flexible bag body, and a filter membrane (233) attached to the second port, and the retaining structure (24) is configured to enable the second port and the filter membrane to move from a first position to a second position, wherein in the first position, the second port and the filter membrane are located above the liquid surface (L) of the culture medium without contacting the culture medium, and in the second position, the second port and the filter membrane are located below the liquid surface of the culture medium. Preferably, the holding mechanism (24) comprises a first magnet member (241) located inside the flexible bag body, a second magnet member (242) located outside the flexible bag body, and an attachment member (243) for the first magnet member; in the first position, the first magnet member and the second magnet member clamp the second port and the filter membrane to the top side wall of the flexible bag body through magnetic force; and when it is necessary to use the fluid exchange pipeline for fluid exchange, the magnetic force on the first magnet member can be eliminated by removing the second magnet member (242), thereby loosening the second port and the filter membrane and causing them to move to the second position under the action of gravity; Preferably, the cell culture bag (2) further comprises an air inlet port (4) and an air exhaust port (5), wherein the air inlet port is used to be in fluid communication with an external gas source, and the air exhaust port is in fluid communication with a pressure control valve (6) located outside the cell culture bag (2). More preferably, an air filter (7) is further provided between the air exhaust port (5) and the pressure control valve, and a heater (8) is provided on the outside of the air filter for heating the gas exhausted from the air exhaust port. Optionally, the pressure control valve (6) is a one-way valve. Preferably, the pore size of the filter membrane (233) is 20 μm to 100 μm; Preferably, it further comprises a temperature sensor (9), a pH sensor and a dissolved oxygen sensor inserted into the cell culture bag (2); more preferably, a flexible sleeve (90) is provided on the outside of the temperature sensor so that the temperature sensor is not in direct contact with the culture medium; more preferably, the attachment (243) is a tether, wherein one end of the tether is connected to the first magnet (241), and the other end is connected to the temperature sensor (9) inserted into the cell culture bag (2); Preferably, the driver is configured to select a suitable rotation rate to drive the reactor tank (1) at different culture stages, wherein in the culture medium equilibrium stage, the rotation rate is selected to be 40 rpm to 50 rpm; in the single cell inoculation stage, the rotation rate is selected to be 40 rpm to 50 rpm; after the liquid replacement is completed, the rotation rate is selected to be 45 rpm to 55 rpm; Preferably, the reactor tank has a tank height, and the second position is located at a height between 1 / 4 and 1 / 3 of the tank height.

2. A stirred bioreactor (10'), comprising: A cylindrical tank body (1') defining a cylindrical chamber (22') for accommodating cell culture; an agitator (2') extending into the cylindrical chamber; and A driving motor (3') for driving the stirrer to rotate; The stirrer comprises a stirring shaft (20'), a first end of which is connected to a driving motor, one or more coaxial stirring paddles (21') are fixedly mounted on the shaft, extending as far as the second end of the stirring shaft, and each stirring paddle independently has the following structure: a cylindrical sleeve (24') fixedly connected to the shaft and having a common central axis with the shaft, and three stirring blades (23') evenly distributed along the outer circumference of the cylindrical sleeve, wherein each stirring blade has an angle of 45° with a plane perpendicular to the central axis, and a ratio of the diameter of the stirring blade to the diameter of the cylindrical chamber is 0.3 to 0.9, Preferably, in the stirred bioreactor, (1) at least one of the stirring paddles has a stirring blade with a diameter of 4 cm; and / or (2) the diameter of the cylindrical chamber is 8 cm; Preferably, the stirred bioreactor comprises a cover (11') for sealing a cylindrical chamber, a liquid inlet pipeline and a liquid outlet pipeline connected to the cylindrical chamber fluid through the cover, and an air inlet port, an air exhaust port and a sampling port arranged in the cover; and also comprises a temperature sensor, a pH sensor and a dissolved oxygen sensor inserted into the cylindrical chamber through the cover, and a sleeve is provided on the outside of the temperature sensor so that the temperature sensor does not directly contact the culture medium.

3. A method for culturing iPSCs, comprising inoculating iPSCs into a culture medium, and using a bioreactor, characterized in that: During the culture process, the culture medium containing the iPSCs moves in a horizontal circular manner, or mainly in a horizontal circular manner, in the bioreactor.

4. The culture method of claim 3, wherein the method comprises the following steps: (a1) adding a predetermined volume of culture medium into the bioreactor; (a2) inoculating iPSCs into culture medium; (a3) causing the culture medium containing the iPSCs to move in a horizontal circular manner, or mainly in a horizontal circular manner, in the bioreactor to culture the iPSCs, Optionally, before step (a1), step (a0) is further included, wherein iPSCs are cultured and expanded in a non-bioreactor manner for subsequent inoculation; preferably, they are artificially cultured in a culture dish or culture bottle in an adherent or suspended manner to obtain at least about 2×10 7 Live cells, for subsequent inoculation, Optionally, after step (a3), a step (a4) of harvesting the cultured iPSCs is also included. Preferably, step (a4) includes: collecting all the aggregate suspension (i.e., the culture medium containing the aggregates) into a receiving bottle connected to the bioreactor, and then dissociating the aggregates into single cells.

5. The method of claim 3 or 4, wherein the bioreactor is the bioreactor of claim 1 or 2.

6. The method of claim 4 or 5, wherein the predetermined volume in step (a1) is at least 200 mL; preferably, at least 300 mL, at least 400 mL, at least 500 mL, at least 600 mL, at least 700 mL, at least 800 mL, at least 900 mL, at least 1000 mL, at least 1100 mL, at least 1200 mL, at least 1300 mL, at least 1400 mL, at least 1500 mL, at least 2000 mL, at least 3000 mL, at least 5000 mL, at least 7500 mL, at least 10000 mL, at least 15000 mL, at least 20000 mL or at least 30000 mL; more preferably, the predetermined volume is in the range of 300 mL-1500 mL; most preferably, the predetermined volume is in the range of 300 mL-600 mL.

7. The method according to any one of claims 3 to 6, wherein the bioreactor is a turbulent bioreactor and its operating conditions include one or more of the following: ① Monitor and maintain the reactor speed at 40-80 rpm; ② Monitor and maintain the culture medium temperature at approximately 37°C; ③ The DO value of the culture system is maintained at 5-80%; ④The pH value is maintained between 6.8 and 7.4, and air is passed when the pH value decreases or is too low, and CO2 gas is passed when the pH value increases or is too high; ⑤ The pressure inside the bag is controlled to about 500Pa through the pressure control valve at the exhaust gas end, so that the bag is inflated, and the exhaust gas is continuously heated by the heater. Preferably, the torrent bioreactor is the torrent bioreactor of claim 1, or The bioreactor is a stirred tank bioreactor and its operating conditions include one or more of the following: ① Monitor and maintain the stirring blade speed at 80-140 rpm; ② Monitor and maintain the culture medium temperature at approximately 37°C; ③ The DO value of the culture system is maintained at 5-80%; ④The pH value is maintained between 6.8 and 7.4, and air is passed when the pH value decreases or is too low, and CO2 gas is passed when the pH value increases or is too high; ⑤The tail gas is discharged after entering the tail gas bottle. Preferably, the turbulent flow bioreactor is the stirred-flow bioreactor of claim 2.

8. The method according to any one of claims 4 to 7, wherein In step (a1), air is continuously passed through the mixture at a flow rate of 30 mL / min to 80 mL / min; In step (a2), air is passed through the mixture at a flow rate of 50 mL / min to 100 mL / min; and / or In step (a3), air is continuously passed through the mixture at a flow rate of 100 mL / min to 300 mL / min.

9. The method according to any one of claims 4 to 8, wherein in step (a2), the cell density in the bioreactor after inoculation is 1.5×10 5 Viable cells / mL and no more than 3.5×10 5 Viable cells / mL, Optionally, the culture medium comprises a basal medium and an additive, wherein the basal medium comprises hPSC-AOF, hPSC-Scale or Essential 8, and the additive comprises ROCKi, such as Y-27632.

10. The method according to any one of claims 4 to 9, wherein the step (a3) ​​further comprises a fluid replacement operation once a day, preferably the fluid replacement does not change or substantially does not change the volume of the culture system, Preferably, The fluid replacement operation includes performing cell fluid replacement through a dedicated fluid replacement pipeline; preferably, the cell fluid replacement includes the following steps: Maintain or preferably appropriately increase the reactor speed; replace 25% to 75%, preferably about 50% of the volume of the culture medium of the culture system through the liquid replacement pipeline; or, The fluid replacement operation includes performing cell fluid replacement through an external cell filter bag; preferably, the cell fluid replacement includes the following steps: pausing the operation of the reactor; extracting the culture fluid into the external cell filter bag, retaining the aggregates by filtering and removing 25% to 75%, preferably about 50% of the volume of the culture system, and then adding a roughly equal volume of fresh culture medium, and finally returning the culture medium after the fluid replacement to the tank, and resuming the operation of the reactor; or, The fluid replacement operation includes using a dual inlet / outlet pipeline as a fluid replacement channel. Preferably, the cell fluid replacement includes the following steps: at least about 5 minutes before the start of the fluid replacement, pausing the movement of the bioreactor to allow the aggregates in the culture medium to settle to the lower half or even the bottom of the culture system; then completing the fluid replacement by extracting the old culture medium from the upper half of the culture system and adding new culture medium, and the fluid replacement volume is 25% to 75% of the volume of the culture system, preferably about 50%.

11. The method according to any one of claims 3 to 10, wherein the iPSCs grow in a manner of forming aggregates.

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