Method for automatically digesting cell aggregates

Abstract

The invention relates to a method for automatically digesting cell aggregates, in particular to a method for digesting cell aggregates through a cell treatment instrument such as a Sepax C-Pro cell treatment instrument, and provides a universal technical platform for realizing large-scale cell treatment.

Description

Technical Field

[0001] This invention belongs to the field of stem cell technology, specifically relating to a method for digesting cell aggregates using a cell processor, which provides a general-purpose technology platform for large-scale cell processing. Background Technology

[0002] Pluripotent stem cells can differentiate into various cells that make up biological tissues. Therefore, in the field of regenerative medicine, research using stem cells can be used to treat diseases or tissue damage.

[0003] One current method for pluripotent stem cell (PSC) suspension culture is to achieve proliferation or differentiation through the formation of cell aggregates. However, in suspension culture, continuous cell proliferation leads to a continuous increase in the size of the cell aggregates. When a certain diameter is exceeded, it becomes impossible to provide sufficient nutrients and / or growth factors or other signaling molecules to the cells within the aggregates, resulting in spontaneous differentiation or apoptosis. Therefore, for continuous expansion or continued directed differentiation, it is necessary to dissociate the cell aggregates and re-seed the resulting single cells ("passaging").

[0004] However, compared to adherent cells, cells cultured in suspension have tighter intercellular connections. During artificial digestion, adherent cells generally only require adding digestive solution to cover the culture surface or gently agitating the cells, but the artificial digestion of aggregates involves more agitation. Secondly, the artificial digestion process involves multiple centrifugation steps and supernatant disposal. Due to the limited processing volume of centrifuges, batch digestion is necessary when processing large quantities. Furthermore, subjective judgment can easily lead to insufficient digestion, over-digestion, or inadequate termination of digestion during the completion and termination of artificial digestion.

[0005] Therefore, with the increase in commercial production of suspended cells, there is an urgent need to develop a new method for digesting cell aggregates to meet the need for dissociating cell aggregates on a larger scale. Summary of the Invention

[0006] This disclosure provides a method for digesting cell aggregates, achieving continuous, stable, and closed automated digestion of cell aggregates. Specifically, the method includes the following steps: optionally concentrating and washing cells in a piston-type centrifuge chamber of a cell processor; dispersing cell aggregates, preferably aggregates linked into spheres, by using a centrifuge chamber incubation mode (pulsating forward and reverse rotation) and the "separation force" generated by the digestion solution; optionally, finally collecting the processed single-cell suspension by using a stop solution and a concentration and washing mode. This method can be applied in the single-cell passage, cryopreservation, or harvesting of cell aggregates. For example, this method can achieve automated digestion of cell aggregates and subsequent continuous passage culture by using a "tandem" reactor. Considering that manual digestion involves multiple container culture and processing operations that lead to a significant increase in cell mass loss during large-volume culture, this method utilizes equipment for cell aggregate digestion, which can effectively replace manual digestion.

[0007] In one aspect, this disclosure provides a method for digesting cell aggregates, comprising the following steps:

[0008] (a) Obtaining cell aggregates;

[0009] (b) Digesting the cell aggregates, wherein the digestion is performed in a cell processing apparatus; and

[0010] (c) Collect the digested cells.

[0011] In some embodiments, the cell processor described in digestion process (b) of this disclosure has a piston-type centrifuge chamber.

[0012] In some embodiments, the digestion process (b) of this disclosure includes causing the cell aggregates to undergo pulsed forward and reverse axial motion in the piston-type centrifuge chamber.

[0013] In some embodiments, the cell processing instrument disclosed herein is Sepax C-Pro, PRO, Cyclone series fully automated cell processors (such as the Mini Cyclone), Gentle Flex Pro fully enclosed large-volume cell processor (Cyclone), TCell-Pro (Dongfulong), with Sepax C-Pro being the best choice.

[0014] In some embodiments, the digestion process of this disclosure includes adding a cell dissociation agent to the cell aggregates, preferably the cell dissociation agent including Accutase, Accumax, trypsin, EDTA, trypsin-EDTA, recombinant trypsin, or TrypLE (such as TrypLE). TM Express, TrypLE TM Select(1X), TrypLE TMSelect(10X)), collagenase, elastase, cellulase, alginate lyase, glucanase, or dispersase.

[0015] In some embodiments, the digestion process of this disclosure further includes adding nucleases to the cell aggregates.

[0016] In some embodiments, the cells disclosed herein include, but are not limited to, totipotent cells, embryonic stem cells (e.g., human embryonic stem cells) and their differentiated cells, induced pluripotent stem cells (iPSCs) and their differentiated cells, pluripotent stem cells, epidermal progenitor cells, mesenchymal stem cells, pancreatic β-cell progenitor cells, pancreatic β-cells, cardiac progenitor cells, cardiomyocytes, hepatic progenitor cells, hepatocytes, muscle cell progenitor cells, muscle cells, kidney cells, osteoblasts, hematopoietic progenitor cells, dental follicle cells, hair follicle cells, retinal pigment epithelial cells, neural stem cells, neurons, astrocytes, oligodendrocytes, microglia, inner ear cells and fibroblasts (e.g., dermal fibroblasts), adult bone marrow stem cells, peripheral blood stem cells, umbilical cord stem cells, placental stem cells, cells of immune system cell types, such as lymphocytes, natural killer cells, macrophages and dendritic cells, preferably induced pluripotent stem cells.

[0017] In some embodiments, the cells disclosed herein include pluripotent stem cells, such as embryonic stem cells (e.g., human embryonic stem cells) and induced pluripotent stem cells (iPSCs).

[0018] In some embodiments, the cells disclosed herein include iPSC-derived cells, such as neural precursor cells (NPC), kidney progenitor cells, cardiac precursor cells, pancreatic precursor cells (PPC), hematopoietic stem / progenitor cells (HSPC), and hematopoietic progenitor cells (HPC).

[0019] In some embodiments, the digestion time range of this disclosure is about 3-60 min, 5-50 min, 6-40 min, 8-35 min, 10-30 min, 11-29 min, 12-28 min, 13-27 min, 14-26 min, 15-26 min, for example about 8 min, about 10 min, or about 25 min.

[0020] In some embodiments, the digestion time of this disclosure is at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 8 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 40 minutes, at least about 50 minutes, or at least about 60 minutes.

[0021] In some embodiments, the cell type is pluripotent stem cells, and the digestion time ranges from about 3 to 20 minutes; preferably about 4 minutes, about 5 minutes, about 6 minutes, about 6-8 minutes, about 8 minutes, about 6-9 minutes, about 9-16 minutes, about 10 minutes, or about 10-15 minutes; most preferably about 5 minutes, about 6-8 minutes, about 8 minutes, about 10 minutes, or 10-15 minutes.

[0022] In some embodiments, the cell type is a pluripotent stem cell-derived cell, and the digestion time is 15-30 min, 17-29 min, 19-28 min, preferably 21-27 min.

[0023] In some embodiments, the cell type is NPC cells, and the digestion time of this disclosure is preferably about 22 min, 23 min, 24 min, 25 min, 26 min, 27 min or 28 min, preferably about 25 min.

[0024] In some embodiments, the digestion process of this disclosure includes applying centrifugal force to the cell aggregates, preferably at least 40g, at least 50g, at least 60g, at least 70g, at least 80g, at least 90g, at least 100g, at least 110g, at least 120g, at least 130g, at least 140g, at least 150g, at least 200g, at least 250g, at least 300g, at least 400g, for example, at least 40-400g, at least 50-300g, at least 60-200g, at least 60-150g, at least 70-100g, at least 75-100g, at least 75-90g, preferably at least 80g.

[0025] In some embodiments, the average particle size of the cell aggregates obtained in step (a) of this disclosure is about 10-5000 μm, 20-4000 μm, 30-3000 μm, 40-2000 μm, 50-2000 μm, 50-1000 μm, 80-1000 μm, 100-1000 μm or 200-900 μm, preferably about 300-800 μm.

[0026] In some embodiments, the cells are pluripotent stem cells, and the average particle size of the obtained cell aggregates is 200-500 μm, 220-480 μm, 230-470 μm, 240-460 μm, 250-450 μm, 260-440 μm, 270-430 μm, 280-430 μm, 290-420 μm, 290-410 μm or 290-400 μm, preferably about 300-400 μm.

[0027] In some embodiments, the cells are pluripotent stem cell-derived cells, and the average particle size of the obtained cell aggregates is 500-900 μm, 510-890 μm, 520-880 μm, 530-870 μm, 540-860 μm, 530-850 μm, or 560-840 μm. In some embodiments, the cell type is NPC cells, and the average particle size of the obtained cell aggregates is 570-830 μm, 580-820 μm, or 590-810 μm, preferably about 600-800 μm.

[0028] In some embodiments, step (a) of this disclosure includes a concentration and / or washing step, preferably performed in a cell processing instrument. In some embodiments, a suitable cell buffer is used in the above concentration and / or washing step, including but not limited to HEPES, sodium bicarbonate, DPBS, or PBS, preferably DPBS, and more preferably CTS. (TM) DPBS(1X).

[0029] In some embodiments, the centrifugal force in the step of obtaining cell aggregates in this disclosure is about 50g, about 60g, about 65g, about 70g, about 75g, about 80g, about 85g, about 90g, about 95g, about 100g, about 110g, about 120g, about 130g, about 140g, about 150g, about 200g, about 300g, about 400g, or about 500g, for example, about 50-500g, about 60-300g, about 60-200g, about 60-150g, about 70-120g, preferably about 100g.

[0030] In some embodiments, step (c) of this disclosure includes collecting the digested cells using a centrifugation method, preferably using at least 50g, at least 100g, at least 200g, at least 300g or at least 400g, for example about 50-600g, for example about 100-500g, about 200-400g, preferably such as 300g.

[0031] In some embodiments, the method of this disclosure includes performing the following procedure: a cell aggregate suspension is subjected to two rounds of infusion, concentration, and one round of washing followed by buffer replacement—the culture medium is replaced with washing buffer—DPBS; digestion solution is infused and pulsed centrifugation is initiated, allowing the cell aggregates to be fully exposed in the digestion solution and continuously vortexed for 5–30 min; a stop solution is infused and a homogenized single-cell suspension is finally harvested after two rounds of washing. The method of this disclosure includes digestion using a bead wash procedure.

[0032] In some embodiments, the method of this disclosure includes the following steps performed in a Sepax C-Pro cell processing instrument: filling a suspension of 400 mL cell aggregate culture system with an average particle size of approximately 250-1000 μm into a 500 mL reservoir bag, and aseptically connecting it to... Figure 3 Pipeline 4; pass ≥230mL of cleaning solution through Figure 3 Connect the extension tube 8 and the tubing 5; pour ≥50 mL of digestion solution (Accutase) into a 100 mL storage bag, and then... Figure 3 Connect the extension tube 8 and the tubing 5; pass ≥400mL of the stop solution through... Figure 3 Connect the extension tube 8 and the pipeline 5, and click the program to start the automatic digestion of the aggregate.

[0033] In some implementations, the method of this disclosure includes using the procedures shown in the following table:

[0034]

[0035] In a second aspect, this disclosure provides the application of the Sepax C-Pro cell processing instrument in digestion processes, preferably the digestion processes comprising cell aggregates.

[0036] In some embodiments, the methods of this disclosure have comparable or higher cell viability compared to artificial digestion. In some embodiments, the cell viability of the methods of this disclosure is at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, and at least 95%, or even 100%, for example, about 80%, 85%, and 90%, compared to artificial digestion.

[0037] In some embodiments, the methods of this disclosure have comparable or higher cell recovery rates compared to artificial digestion. In some embodiments, the cell recovery rates of the methods of this disclosure are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95%, or even 100%, compared to artificial digestion, for example, about 74%, 94%, 97%, and 100%.

[0038] In some embodiments, the method of this disclosure allows for the continued culture of single cells after digestion to achieve culture results substantially consistent with those of artificial digestion, and / or without causing excessive shear stress damage to the aggregates.

[0039] In some implementations, after single cells digested by the method of this disclosure are further cultured, the particle size of the cell aggregates formed is basically consistent with that of artificially digested cells.

[0040] In some embodiments, the iPSC aggregates digested by the methods of this disclosure have pluripotency-related markers, such as TRA-1-60, SSEA4, NANOG, or OCT4. In some embodiments, the cells digested by the methods of this disclosure have about 70-99%, preferably 90-98%, more preferably 95.44% TRA-1-60. In some embodiments, the cells digested by the methods of this disclosure have about 70-99%, preferably 95-99%, more preferably 98.96% SSEA4. In some embodiments, the cells digested by the methods of this disclosure have about 70-99%, preferably 93-98%, more preferably 95.20% NANOG. In some embodiments, the cells digested by the methods of this disclosure have about 70-99%, preferably 93-98%, more preferably 96.49% OCT4. In some embodiments, the cells digested by the methods of this disclosure have approximately 95.44% TRA-1-6, approximately 98.96% SSEA4, approximately 95.20% NANOG, and approximately 96.49% OCT4.

[0041] In some embodiments, the NPC aggregates digested by the methods of this disclosure have neural progenitor cell-related markers, such as PAX6 and SOX2. In some embodiments, the NPC aggregates digested by the methods of this disclosure are OCT4 negative.

[0042] The term "aggregate" in this disclosure can be used interchangeably with "cell aggregate," referring to a clump formed by a collection of cells adhered to each other. Cell aggregates include cell groups, embryoid bodies, spheres, spheroids, and organoids. Preferably, in cell aggregates, cells adhere to each other surface-to-surface. In some embodiments, cells adhere to each other in part or all of the cell aggregate, for example, forming adhesion junctions. In some embodiments, two or more cell aggregates may be further artificially adhered or aggregated. Cell aggregates also contain clumps of cells for further adhesion or aggregation, as well as as assemblages. The morphology of cell aggregates is not limited to spherical shapes; for example, they can be dispheres, beaded structures, aggregates of spheres, rope-like structures, and branched structures.

[0043] In this article, "suspension culture" refers to a culture system or method in which cells, single cells, cell aggregates, or mixtures of single cells and aggregates are suspended in the culture medium rather than adhered to the surface. In a liquid culture medium, under constant agitation, the cultured cells expand and grow in three dimensions in a non-adherent manner, either as single cells or forming small cell clusters. Especially for the latter, although they are actually in suspension, the adhesion between cells within the cell cluster provides a state similar to "adherence," and the extracellular matrix (ECM) between the cells in the cell cluster also meets the needs of cell growth. Such cell clusters can be called "aggregates."

[0044] The term "culture medium" as used in this article refers to a nutrient solution used for the culture, growth, or proliferation of cells.

[0045] The terms "digestion" and "digestion treatment" used in this disclosure are used interchangeably and refer to the process of reducing the adhesion between some or all cells in a cell aggregate. In some embodiments, digestion can disperse some or all cells in a cell aggregate into a single-cell state where they are not adhered to each other.

[0046] The "cell processing instrument" described in this disclosure refers to high-end equipment used in the production of cell therapy products, which typically possesses multiple functions including: aseptic operation, automation, scalability, data tracking and recording, flexibility and customizability, and reduced production costs. Among these, the aseptic operation function means that the equipment design ensures that the entire cell processing process is carried out in a sterile environment, reducing the risk of contamination; the automation function means that it can automatically complete a series of operations such as cell separation, transduction, and collection (under appropriate programs); and the scalability means that some system designs take into account the scalability of production scale, and can meet greater production needs by adding equipment units.

[0047] In some embodiments, the "cell processor" can operate under corresponding programs, such as automated digestion programs, passage programs, bead wash programs, etc., preferably programs that are manually set, such as manually setting program parameters. In some embodiments, the "cell processor" can be connected in series or parallel with other systems, preferably in series. In some embodiments, the "cell processor" can be connected to cell culture devices for producing cell aggregates, and / or to other kits (such as one or more additional containers for additive solutions) to perform processes such as concentration, purification, separation, density gradient centrifugation, dilution, volume adjustment, and transduction of cells or cell aggregates.

[0048] In some implementations, the "cell processing apparatus" includes a set of containers for receiving the biological fluid to be separated and the separated components, and optionally one or more additional containers for additive solutions. The hollow centrifugal processing chamber is rotatable about a rotation axis through engagement of the processing chamber with a rotary drive unit. The processing chamber has an axial inlet / outlet for the biological fluid to be processed and the processed components of the biological fluid. This inlet / outlet leads to a variable-volume separation space in which the entire centrifugation of the biological fluid is performed. The processing chamber includes a generally cylindrical wall extending from an end wall of the processing chamber, defining a hollow processing chamber therein that occupies a hollow, open cylindrical space coaxial with the rotation axis. The axial inlet / outlet disposed in the end wall is coaxial with the generally cylindrical wall to open into the hollow processing chamber. The processing chamber contains an axially movable member, such as a piston, within the generally cylindrical wall. A variable-volume separation space is defined in the upper part of the processing chamber by a generally cylindrical wall and an axially movable member contained within the generally cylindrical wall of the processing chamber. Axial movement of the movable member changes the volume of the separation space. The movable member is axially movable within the processing chamber to draw a selected amount of biofluid to be processed into the separation space via the inlet before or during centrifugation, and to expel the processed biofluid components from the separation space via the outlet during or after centrifugation. In some embodiments, the cell processing apparatus of this disclosure may be the system described at the following link: https: / / www.cytivalifesciences.com.cn / zh / cn / solutions / cell-therapy / products-and-technology / autologous-car-t-workflow / sepax-c-pro-cell-processing-system.

[0049] The "piston-type centrifuge chamber" described in this disclosure houses an axially movable component, such as a piston, within its cylindrical walls. This component defines a separation space with a variable volume. The component is axially movable to change the volume of the separation space within the processing chamber, allowing biological fluid to enter or exit the separation space. The piston-type centrifuge chamber can rotate about a rotation axis through engagement of the processing chamber with a rotary drive unit.

[0050] The "countercurrent centrifugation" described in this disclosure is a general method for separating cells based on cell deposition characteristics (size and density). Cells are suspended in a fluidized bed by applying a constant flow force opposite to the centrifugal force; the suspended cells are gently concentrated without forming a precipitate in the cone, and then effectively washed; by adjusting the centrifugation speed and flow rate to create a force imbalance, cell types of different sizes and densities are separated. For example, the Rotea system can be used for countercurrent centrifugation to separate cells.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods of this disclosure pertain. Attached Figure Description

[0052] The preferred embodiments of the invention described in the following detailed description will be better understood when read in conjunction with the accompanying drawings. The drawings show presently preferred embodiments for illustrative purposes. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the drawings.

[0053] Figure 1A and 1B The flowchart of cell aggregate digestion is shown, in which, Figure 1A A flowchart of artificial digestion is shown; Figure 1B The flowchart of the automatic digestion process is displayed.

[0054] Figure 2 The bright-field plot of iPSC aggregates after autodigestion by Sepax C-Pro is shown.

[0055] Figure 3The Sepax C-Pro cell processing pipeline diagram is shown, where each label represents the following: 1. Chamber: 220mL total volume; 2. Pressure detector; 3. Rotary valve branch: Three automatically controlled rotary valves that guide fluid flow, automatically controlling the direction of liquid flow in the pipeline after being programmed; 4. Initial bagging pipeline: Inflow pipeline for aggregate suspension; 5. Washing solution pipeline: In addition to cell washing solution CTS-DPBS, digestion solution and termination solution enter through this pipeline; 6. Waste bag: Storage bag for collecting waste liquid from the entire process; 7. Final product pipeline: Pipeline connecting to the storage bag for collecting final product; 8. Dual-tube extension pipeline: Expanding the number of pipelines by connecting pipelines; 9. Single-tube extension pipeline: Extending the length of the pipeline by connecting pipelines.

[0056] Figure 4 The image shows the growth pattern of iPSC aggregates (iPSCs obtained by automatic digestion with Sepax C-Pro).

[0057] Figure 5 The growth map of iPSC aggregates is shown (iPSCs obtained by Rotea autodigestion).

[0058] Figure 6 The particle size distribution of iPSC aggregates is shown (iPSCs obtained by automatic digestion with Sepax C-Pro).

[0059] Figure 7 The particle size distribution of iPSC aggregates is shown (iPSCs obtained from Rotea autodigestion).

[0060] Figure 8 The image shows a bright field plot after the NPC aggregate Sepax C-Pro is automatically digested.

[0061] Figure 9 The Q-PCR diagram of NPC aggregates after autodigestion with Sepax C-Pro is shown.

[0062] Figure 10 This shows the Rotea cell processing pipeline diagram. Example

[0063] Experimental materials

[0064] The instrument and equipment information involved in the embodiments is shown in Table 1 below.

[0065] Table 1

[0066]

[0067] Information on reagents and consumables involved in the embodiments is shown in Table 2 below.

[0068] Table 2

[0069]

[0070]

[0071] Note: The termination solution used during digestion termination is the same as the culture medium used for aggregate culture.

[0072] Example 1: Exploring Key Parameters for Automatic Digestion

[0073] 1.1 Digestion Process

[0074] The specific process of artificial digestion is as follows: Figure 1A As shown.

[0075] The automatic digestion process is as follows Figure 1B As shown. Specifically, the operational logic of automatic digestion is basically the same as that of artificial digestion; however, automatic digestion differs significantly from artificial digestion in the "digestion aggregate" step, namely, in automatic digestion, the entire digestion system undergoes pulsed forward and reverse horizontal rotation, wherein the pulsed forward and reverse horizontal rotation consists of intermittent forward horizontal circular motion, stopping, reverse horizontal circular motion, and stopping during centrifugation.

[0076] iPSC aggregate culture process: Human induced pluripotent stem cells (iPSCs derived from human cells are established by introducing two factors, OCT4 and NANOG, and three small molecules into human fibroblasts, see CN113454230B for details) are cultured at a rate of 6 × 10⁻⁶. 4 Cells were seeded at a density of 300-500 mL into a PBS bioreactor. Day 0 was the day of seeding. From Day 3 to 5, samples were taken daily for observation and medium replacement at a 50% volume ratio. On Day 5-6, when the average aggregate size reached 300-400 μm, the aggregate suspension was harvested (for passage or cryopreservation). The culture medium used throughout was hPSC-AOF, and 10 μM Rocki was added on the day of each passage.

[0077] In the digestion of aggregates, the centrifugal force used in automated digestion is greater than that used in manual digestion, ranging from 60 to 150 g.

[0078] For iPSC aggregates, the centrifugal force for the automated digestion experimental design (using Accutase for 8 min) is shown in Table 3A below.

[0079] Table 3A

[0080] Group Collect aggregates (concentrate and prewash). Digestive aggregates Collect single cells (terminate and wash afterward). Group 1 100g 60g 200g Group 2 100 g 80g 200g Group 3 100g 150g 200g Group 4 100 g 80g 300g Group 5 200 g 80g 300g

[0081] In the digestion step, the digestion time for automated digestion is the same as that for manual digestion. Although the digestion time varies significantly for different digestion solutions, the range for automated digestion is 5–15 min. Specifically, for iPSC aggregates, the digestion times for different digestion solutions in automated digestion are shown in Table 3B below.

[0082] Table 3B

[0083]

[0084]

[0085] 1.2 Investigation of centrifugal force in the digestion of aggregates

[0086] Based on the bead wash program of the Sepax C-Pro cell processor, an automated digestion experiment was conducted according to the design of Group 1, Group 2 and Group 3. After digestion, samples were taken from the final product bag, and artificial digestion was set up as a control group.

[0087] The results are as follows Figure 2 As shown, the cell suspension in group 2 was digested into single cells, with no spherical or clumpy structures or flocculent matter present, indicating that a centrifugal force of 80g was appropriate during the digestion of aggregates. The presence of spherical or clumpy structures in the cell suspension in group 1 indicated that a centrifugal force of 60g was too small and could not completely dissociate the cell aggregates. The presence of significant flocculent matter in the supernatant of group 3 indicated that a centrifugal force of 150g was too large and would lead to over-digestion of cell aggregates.

[0088] 1.3 Study on centrifugal force during pretreatment and posttreatment of digested aggregates

[0089] Waste liquid was collected and cell loss was analyzed during the concentration, prewash, termination and postwash stages of cell processing. Waste-1, waste-2, waste-3 and waste-4 correspond to the waste liquid bags of each of the above stages.

[0090] The results showed that almost no cell spheres were present in waste-1 and waste-2 bags in group 2, while a small amount of single-cell suspension was present in waste-3 and waste-4, with a cell recovery rate of 74%. Increasing the centrifugal force for collecting single cells to 300g (groups 4 and 5) resulted in cell recovery rates of 97% and 94%, respectively. However, a small amount of flocculent material was present in waste-1 bag in group 5, suggesting that a centrifugal force of 200g for collecting aggregates caused some damage to aggregate collection.

[0091] In summary, the optimal centrifugal force parameters for collecting aggregates, digesting aggregates, and collecting single cells are 100g, 80g, and 300g, respectively.

[0092] Example 2: Automated digestion of human induced pluripotent stem cell (iPSC) aggregates

[0093] 2.1 Experimental Methods

[0094] Cell culture process: iPSCs (human cell-derived iPSCs were established by introducing two factors, OCT4 and NANOG, and three small molecules into human fibroblasts, see CN113454230B for details) were cultured at 6×10⁻⁶. 4 Cells were seeded at a density of 300-500 mL into a PBS bioreactor. Day 0 was the day of seeding. From Day 3 to 5, samples were taken daily for observation and medium replacement at a 50% volume ratio. On Day 5-6, when the average aggregate size reached 300-400 μm, the aggregate suspension was harvested (for passage or cryopreservation). The culture medium used throughout was hPSC-AOF, and 10 μM Rocki was added on the day of each passage.

[0095] Each passage procedure: Take a 1-2 mL sample of the aggregate suspension and photograph it under a microscope. If the average particle size of the aggregate is determined to be 300-400 μm, transfer a certain volume of the aggregate suspension to centrifuge tubes and storage bags for artificial and automatic digestion, respectively.

[0096] Artificial digestion

[0097] For artificial digestion, taking a 50 mL aggregate suspension as an example, the aggregates in the centrifuge tube are first allowed to settle for 3 minutes, then the supernatant is discarded. DPBS is added to a total volume of about 50 mL, and the mixture is allowed to settle for another 3-10 minutes, then the supernatant is discarded. 2-4 times diluted dissociation buffer (Accumax) is added to a total volume of 10 mL, and dissociation is carried out for 5-15 minutes. During this period, the centrifuge tube is shaken by hand every 2-3 minutes to promote the dissociation of aggregates. After the aggregates are dissociated into a homogeneous single-cell suspension, culture medium is added to stop the process and harvest the aggregates.

[0098] Sepax C-Pro automatic digestion

[0099] Sepax C-Pro automated digestion program: Pour a suspension of 400 mL of iPSC aggregate culture system with an average particle size of 300-400 μm into a 500 mL reservoir bag and aseptically connect to... Figure 3 Pipeline 4 (aseptic connection and sealing throughout the entire process); pass ≥230mL of cleaning solution through Figure 3 Connect extension tube 8 and tubing 5; pour ≥50 mL of digestion solution (Accutase) and nuclease into a 100 mL storage bag, and then... Figure 3 Connect the extension tube 8 and the tubing 5; pass ≥400mL of the stop solution through... Figure 3Connect the extension tube 8 and the tubing 5; use the "bead wash" program preset in Table 4 to start the automatic digestion of aggregates: that is, after the aggregate suspension passes through 2 rounds of infusion, concentration and 1 round of pre-wash, the culture medium is replaced with washing solution DPBS buffer, digestion solution is infused and pulsed forward and reverse axial motion is started, the aggregates are fully exposed in the digestion solution and continuously shaken and mixed, the digestion time is 10 min (Table 4 Incubation time); stop solution is infused and after 2 rounds of post-wash, a homogenized single-cell suspension is finally harvested.

[0100] Specifically, the Sepax C-Pro automated digestion program for iPSC aggregates is shown in Table 4.

[0101] Table 4. Sepax C-Pro Automated Digestion Program for iPSC Aggregates (Key Operating Parameters)

[0102]

[0103] Rotea Automatic Digestion Program

[0104] Rotea's automated digestion program: The operating parameters for automated digestion have been optimized and determined based on the characteristics of the equipment itself. Except for differences in the logic and structure of the automated digestion equipment, and the differences in the cell culture medium and stop solution, the process flow and reagents (such as digestion solution and washing solution) are consistent with Sepax C-Pro. Specific setup procedures and piping structures are as follows: Figure 10 As shown in the diagram, the aggregate suspension is continuously fed in while centrifuging to accumulate at the bottom of the centrifuge chamber; the culture supernatant is extracted from the centrifuge chamber through the bottom channel and gradually replaced by the buffer solution—DPBS; the digestion solution (Accutase) is fed in and the digestion solution in the centrifuge chamber and tubing circulates alternately in both directions; the aggregates are fully exposed and dissociated into single cells in the digestion solution, and the digestion time is 8 min (Table 5 Step 11); the stop solution is fed in and the single-cell suspension accumulates again at the bottom of the centrifuge chamber while the digestion solution is gradually and completely extracted from the centrifuge chamber and replaced by the stop solution, finally harvesting the final homogenized single-cell suspension.

[0105] Specifically, the Rotea automated digestion procedure for iPSC aggregates is shown in Table 5.

[0106] Table 5. iPSC aggregate Rotea automated digestion program (key operating parameters)

[0107]

[0108]

[0109] 2.2 Experimental Results

[0110] 2.2.1 Cell viability detection

[0111] The viability of the single-cell suspension obtained after digestion in Example 2.1 was tested. The viability of the Sepax C-Pro experimental group, the Rotea experimental group, and the artificial digestion control group were 85.56%, 72.36%, and 87.21%, respectively.

[0112] 2.2.2 Cell growth results

[0113] The single-cell suspensions digested in the automated digestion experimental group and the artificial digestion control group in Example 2.1 were inoculated (Day 0). On Day 4, the cell aggregates were photographed under a microscope at harvest, and the results are as follows: Figure 4 and Figure 5 As shown.

[0114] like Figure 4 As shown, the Sepax C-Pro automated digestion experimental group (represented by Sepax C-Pro in the figure) and the artificial digestion control group (represented by control in the figure) showed essentially the same cell size and cell viability at Day 4 harvest (represented by Viability in the figure, 83.42% and 81.07%, respectively). Furthermore, the Sepax C-Pro automated digestion experimental group and the artificial digestion control group also showed essentially the same edge compactness, morphological roundness, and single-cell residue at Day 4 harvest (almost no free single cells, flocculent material, or cell clumps were present).

[0115] In addition, such as Figure 5 As shown, the experimental group (represented by Rotea in the figure) that underwent automated digestion using the Rotea cell processor based on the countercurrent centrifugation principle, compared to the control group (represented by control in the figure), exhibited significantly smaller aggregate sizes and more aggregate damage at harvest on Day 4 after inoculation. Furthermore, the viability of cell aggregates obtained from the Rotea automated digestion experimental group and the control group underwent automated digestion were 69.2% and 89.2%, respectively. That is, compared to the control group under automated digestion, the cell aggregates in the Rotea automated digestion experimental group showed poor growth and lower cell viability.

[0116] The above results indicate that cells processed by automatic digestion using a cell processor with a piston centrifuge chamber can achieve culture results that are basically the same as those obtained by traditional manual digestion. Therefore, digestion using a cell processor with a piston centrifuge chamber will not cause excessive shear force damage to the aggregates.

[0117] 2.2.3 Particle size analysis

[0118] The cell growth images harvested on Day 4 in Example 2.2.2 were compiled, and the aggregate diameter and particle size uniformity were analyzed using ImageJ. Specifically, the aggregate diameter data detected in each whole cell image were summarized, and the percentage of aggregates in different particle size ranges was calculated to generate corresponding curves, such as... Figure 6 and Figure 7 As shown.

[0119] The average particle size statistics of the cell aggregates harvested on Day 4 after continued culture of the Sepax C-Pro automated digestion experimental group (represented by Cytiva in the figure) and the artificial digestion control group (represented by control in the figure) are shown in the figure. Figure 6 See Table 6. It can be seen that after further culture using the Sepax C-Pro cell processor with a piston-type centrifuge chamber, the particle size of the harvested cell aggregates was basically consistent with that of those obtained through artificial digestion. Furthermore, statistical data on the average particle size of cell aggregates harvested on Day 4 of further culture using the Rotea automated digestion experimental group (based on countercurrent centrifugation) and the artificial digestion control group are shown in Table 6. Figure 7 See Table 7. It can be seen that after further culturing the digested aggregates in the Rotea automated digestion experimental group, the particle size of the harvested cell aggregates differed significantly from that of those from artificial digestion.

[0120] It is evident that the single cells formed after treatment with Sepax C-Pro automated digestion showed better growth ability, while the single cells formed after treatment with Rotea automated digestion showed poorer growth ability due to damage to the cells caused by the equipment.

[0121] Table 6

[0122] Grouping Average diameter (μm) Control 426.6 Sepax C-Pro 430.9

[0123] Table 7

[0124] Grouping Average diameter (μm) Control 363.5 Rotea 230.8

[0125] 2.2.4 Multiplier Analysis

[0126] 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., 1.5 × 10⁻⁶. 5 Cells / mL.

[0127] The results showed that the cells harvested from the automatic digestion experimental group (i.e., the Sepax C-Pro automatic digestion experimental group) and the artificial digestion control group by the piston centrifuge chamber cell processor increased by 8.1 and 8.3 times, respectively. In other words, the cell doubling calculated from the cell number harvested by the automatic digestion of the piston centrifuge chamber cell processor and the artificial digestion was basically the same.

[0128] In addition, the number of cells harvested from the Rotea automated digestion experimental group and the artificial digestion control group increased by 6.1 and 8.2, respectively.

[0129] 2.2.5 FACS Result Analysis

[0130] The pluripotency-related markers of iPSC aggregates, OCT4 (BD#560186), NANOG (BD#560483), TRA-1-60 (BD#560193), and SSEA4 (BD#560128), were detected by flow cytometry.

[0131] The results are shown in Table 8. The Sepax C-Pro automated digestion group (which used the Sepax C-Pro cell processing instrument) showed basically normal results in the four pluripotency indicators of iPSC aggregates that continued to be cultured in shake flasks (i.e., the positive cell ratios of the four cell pluripotency-related markers TRA-1-60, SSEA4, NANOG and OCT4), and were basically consistent with the artificial digestion group (Table 8).

[0132] This indicates that automated digestion using the Sepax C-Pro cell processor does not affect the pluripotency of iPSCs.

[0133] Table 8

[0134]

[0135] Example 3: Automatic digestion of NPC aggregates

[0136] Based on the automated digestion of iPSC aggregates, we optimized the processing time and digestion solution formulation, and developed an automated digestion process for iPSC-derived cells, namely neural progenitor cells (NPC) aggregates.

[0137] The culture process for NPC aggregates is based on patent CN113604434A. The procedure for each subculture is as follows: Take a 1-2 mL sample of the aggregate suspension and photograph it under a microscope. If the average particle size of the aggregates is determined to be 600-800 μm, transfer a certain volume of the aggregate suspension to centrifuge tubes and storage bags for manual and automatic digestion, respectively.

[0138] Artificial digestion

[0139] Taking a 50 mL aggregate suspension as an example, the aggregates in the centrifuge tube should be allowed to settle for 3 minutes, then the supernatant should be discarded. Add DPBS to the total volume of about 50 mL, allow to settle for another 3-10 minutes, then discard the supernatant. Add 1-2 times diluted dissociation buffer (Accumax) to the total volume of 10 mL, and dissociate for 10-20 minutes. During this period, shake the centrifuge tube by hand every 2-3 minutes to promote the dissociation of the aggregates. After the aggregates are dissociated into a homogeneous single-cell suspension, add culture medium to stop the process and harvest.

[0140] Sepax C-Pro automatic digestion

[0141] In Sepax C-Pro, the average particle size of the aggregates before digestion was 700 μm, the digestion solution was Accumax, and the digestion time was 25 min. The digestion program is shown in Table 9 below.

[0142] Table 9. NPC Aggregate Sepax C-Pro Automated Digestion Procedure

[0143]

[0144] Results: Cell images after digestion of NPC aggregates showed that, regardless of Sepax C-Pro Automatic digestion In both the experimental and control groups, most aggregates were dissociated and formed into single cells or small cell clusters. A small number of large cell clusters remained at the bottom of the single-cell suspension collection bags (which could be removed later using a cell filter). Figure 8 Where ctl represents the artificial control group; c-Pro represents Sepax C-Pro. Automatic digestion (Experimental group). Furthermore, the recovery rate = 20 mL of cells harvested via automated digestion / 20 mL of cells harvested via manual digestion = 100%, meaning the recovery rates of NPC aggregates were consistent between automated and manual digestion using Sepax C-Pro. The cell viability after single-cell suspension seeding and continued culture until Day 4 was 85.20% in the Sepax C-Pro group and 86.79% in the manual group, indicating that digestion of NPC aggregates using the Sepax C-Pro cell processor did not reduce cell viability.

[0145] The relevant biomarkers of cellular mRNA levels were detected using appropriate kits and methods. Results are as follows: Figure 9 As shown, after digesting NPC aggregates in the Sepax C-Pro automated digestion group and the artificial digestion group, the expression of relevant markers (PAX6, SOX2, and OCT4) was basically the same. Figure 9 Among them, the NPC markers PAX6 and SOX2 were positive, while the iPSC marker OCT4 was negative.

Claims

1. A method for digesting cell aggregates, comprising the following steps: (a) Obtaining cell aggregates; (b) Digesting the cell aggregates, wherein the digestion is performed in a cell processing apparatus; and (c) Collect the digested cells.

2. The method of claim 1, wherein the cell processor has a piston-type centrifuge chamber.

3. The method of claim 2, wherein the digestion process comprises causing the cell aggregates to undergo pulsed forward and reverse axial motion in the piston centrifuge chamber.

4. The method of any one of the preceding claims, wherein the cell processing instrument is Sepax C-Pro, Cyclone, Gentle Flex Pro fully enclosed large-volume cell processing instrument (Saibridge), TCell-Pro (Dongfulong) or PRO.

5. The method of any of the preceding claims, wherein the cell processor is Sepax C-Pro.

6. The method of any of the preceding claims, wherein the digestion process comprises adding a cell dissociation agent to the cell aggregate, preferably the cell dissociation agent comprising Accutase, Accumax, trypsin, or TrypLE (such as TrypLE). TM Express, TrypLE TM Select(1X), TrypLE TM Select(10X)), preferably Accutase or Accumax.

7. The method of any of the preceding claims, wherein the digestion time is about 3-60 min, 5-50 min, 6-40 min, 8-35 min, 10-30 min, 11-29 min, 12-28 min, 13-27 min, 14-26 min, 15-26 min, for example about 8 min, about 10 min, or about 25 min.

8. The method of any of the preceding claims, wherein the cells include, but are not limited to, totipotent cells, embryonic stem cells (e.g., human embryonic stem cells) and their differentiated cells, induced pluripotent stem cells (iPSCs) and their differentiated cells, pluripotent stem cells, epidermal progenitor cells, mesenchymal stem cells, pancreatic β-cell progenitor cells, pancreatic β-cells, cardiac progenitor cells, cardiomyocytes, hepatic progenitor cells, hepatocytes, muscle cell progenitor cells, muscle cells, kidney cells, osteoblasts, hematopoietic progenitor cells, dental follicle cells, hair follicle cells, retinal pigment epithelial cells, neural stem cells, neurons, astrocytes, oligodendrocytes, microglia, inner ear cells and fibroblasts (e.g., dermal fibroblasts), adult bone marrow stem cells, peripheral blood stem cells, umbilical cord stem cells, placental stem cells, and cells of immune system cell types, such as lymphocytes, natural killer cells, macrophages and dendritic cells.

9. The method of any of the preceding claims, wherein the cells are pluripotent stem cells, and the digestion treatment time is about 3-20 min; preferably about 4 min, about 5 min, about 6 min, about 6-8 min, about 8 min, about 6-9 min, about 9-16 min, about 10 min, or about 10-15 min; most preferably about 5 min, about 6-8 min, about 8 min, about 10 min, or 10-15 min.

10. The method of any of the preceding claims, wherein the cells are pluripotent stem cell-derived cells, and the digestion treatment time is about 15-30 min, 17-29 min, 19-28 min, preferably 21-27 min.

11. The method of any one of claims, wherein the cell aggregates are obtained by suspension culture.

12. The method of any one of claims, wherein step (a) obtains a centrifugal force in the cell aggregate of about 50 g, about 60 g, about 65 g, about 70 g, about 75 g, about 80 g, about 85 g, about 90 g, about 95 g, about 100 g, about 110 g, about 120 g, about 130 g, about 140 g, about 150 g, about 200 g, about 300 g, about 400 g, about 500 g, for example about 50-500 g, about 60-300 g, about 60-200 g, about 60-150 g, about 70-120 g, preferably about 100 g.

13. The method of any claim, wherein step (b) of digesting the cell aggregates comprises applying centrifugal force to the cell aggregates, preferably the centrifugal force being at least about 40g, at least 50g, at least 60g, at least 70g, at least 80g, at least 90g, at least 100g, at least 110g, at least 120g, at least 130g, at least 140g, at least 150g, at least 200g, at least 250g, at least 300g, at least 400g, for example about 40-400g, about 50-300g, about 60-200g, about 60-150g, about 70-100g, about 75-100g, about 75-90g, preferably about 80g.

14. The method of any one of the claims, wherein step (c) includes collecting the digested cells by centrifugation, preferably using at least 50g, at least 100g, at least 200g, at least 300g or at least 400g, for example about 50-1000g, for example about 100-500g, about 200-400g, preferably such as 300g.

15. Application of Sepax C-Pro cell processing instrument in digestion processing.

Images