Domestication composition suitable for Chinese hamster ovary cell CHO-K1, domestication method and application of domestication composition

Abstract

The invention provides a domestication culture medium composition suitable for Chinese hamster ovary cells CHO-K1, a domestication method and application of the domestication culture medium composition. Wherein the composition for domesticating the CHO-K1 comprises a yeast hydrolysate and L-glutamine; wherein the content of the yeast hydrolysate is 0.6 to 2wt%, and the content of the L-glutamine is 0.04 to 0.25 wt%. The composition provided by the invention can realize rapid and efficient CHO-K1 suspension domestication through the yeast hydrolysate and L-glutamine with specific composition, and the domesticated cells are high in viability, high in proliferation speed, single in dispersion, uniform in size, round and transparent.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to an acclimation composition, an acclimation method and applications thereof suitable for Chinese hamster ovary cells CHO-K1. Background Art

[0002] Chinese Hamster Ovary (CHO) cells play a crucial role in the production of biologics. These cells are widely regarded for their well-defined genetic characteristics, low risk of human viral transmission, and excellent safety profile. CHO cells not only demonstrate rapid growth and high-density cultivation in serum-free suspension culture conditions, but also excel in gene amplification and expression, significantly enhancing drug quality control and large-scale production efficiency.

[0003] Another significant advantage of CHO cells is that they produce almost no endogenous proteins, which greatly simplifies the isolation and purification process of target recombinant antibodies. More importantly, CHO cells are able to perform precise post-translational modifications on expressed proteins, such as glycosylation and sialylation, making the produced proteins closer in structure and function to biological proteins naturally produced by the human body. In suspension culture, CHO cells not only grow rapidly, but also perform well in protein expression yield, making them a key expression system for the production of protein-based biological products. CHO cells are highly favored in the biopharmaceutical industry and have become the preferred platform for the production of therapeutic antibodies and other recombinant proteins.

[0004] Chinese hamster ovary K1 (CHO-K1) cells are well-known for their widespread use in recombinant protein production. However, these cells rely on fetal bovine serum (FBS) for adherent culture, which has a complex composition, significant batch-to-batch variability, and the risk of contamination by mycoplasma, viruses, prions, fungi and yeasts, endotoxins, and is also expensive. In addition, FBS contains a variety of proteins, which increases the difficulty of product separation and purification. More importantly, the adherent growth characteristics of CHO-K1 cells have encountered challenges in large-scale production because they need to grow on a surface, which limits the capacity and production efficiency of bioreactors. In order to meet the needs of industrial large-scale production, the ideal cell line should have the ability to be cultured in suspension, which helps to achieve process amplification and improve production efficiency.

[0005] The transition of adherent CHO-K1 cells to serum-free, high-density suspension culture is primarily achieved through direct acclimation and gradient acclimation. Direct acclimation is favored for its ease of operation, but the drastic change in growth environment from adherent to suspension often leads to decreased cell viability, severe clumping, and slower growth, significantly increasing the risk of direct acclimation failure.

[0006] The gradient acclimation method gradually adapts CHO-K1 cells to serum-free suspension culture conditions by gradually adjusting the growth environment. This method allows cells to adapt to new culture conditions in a relatively gentle transition, thereby reducing the cellular stress response caused by environmental abrupt changes. However, this process involves continuously reducing the serum concentration in the culture medium, which not only increases operational complexity but also requires frequent monitoring and adjustments, making the entire acclimation cycle relatively long.

[0007] Therefore, how to provide a CHO-K1 acclimation method with short acclimation time, high success rate and good effect to obtain a single dispersed, uniformly sized, round and transparent suspension cell line with high viability is a technical problem that needs to be solved urgently. Summary of the Invention

[0008] In response to the shortcomings of the direct acclimation method and the gradient acclimation method, the present application provides an acclimation composition, acclimation method and application thereof suitable for Chinese hamster ovary cells CHO-K1.

[0009] Specifically, this application involves the following aspects:

[0010] 1. A composition for acclimating CHO-K1, characterized by comprising yeast hydrolysate and L-glutamine; wherein the content of the yeast hydrolysate is 0.6-2wt%, and the content of the L-glutamine is 0.04-0.25wt%.

[0011] 2. The composition for acclimating CHO-K1 according to item 1, further comprising a basal serum-free medium.

[0012] 3. The composition for acclimating CHO-K1 according to item 2, wherein the basal serum-free medium is one or a combination of two of HyperCHO growth medium and Maibang growth medium.

[0013] 4. The composition for acclimating CHO-K1 according to any one of items 1 to 3, wherein the acclimated CHO-K1 is individually dispersed, uniform in size, round and translucent.

[0014] 5. A method for acclimating CHO-K1, comprising using the composition for acclimating CHO-K1 according to any one of items 1 to 4.

[0015] 6. The acclimation method according to item 5, wherein the acclimation method comprises the following steps:

[0016] 1) Subculturing the revived CHO-K1 cells to restore the state of CHO-K1;

[0017] 2) The recovered CHO-K1 cells are inoculated into the composition for acclimating CHO-K1 and subcultured to acclimate CHO-K1.

[0018] 7. The acclimation method according to item 6, wherein the recovered CHO-K1 is heated to 0.3 to 0.5×10 6 cells / mL were inoculated into the composition for acclimating CHO-K1.

[0019] 8. The acclimation method according to item 6, wherein the total time from the start of the CHO-K1 cell viability being greater than or equal to 95% for three consecutive generations is less than or equal to 25 days, and the doubling time of CHO-K1 is less than or equal to 40 hours when acclimation is completed.

[0020] 9. The acclimation method according to item 6, wherein the acclimated CHO-K1 cells are individually dispersed, uniform in size, round and translucent.

[0021] 10. Use of the composition according to any one of items 1 to 4 in acclimating CHO-K1.

[0022] Beneficial effects of this application

[0023] The composition of the present application includes a yeast hydrolyzate and L-glutamine of a specific composition. When used to acclimate CHO-K1 cells, the total time from the start of the CHO-K1 cell viability to 95% or greater for three consecutive generations is less than or equal to 25 days, and the CHO-K1 doubling time is less than or equal to 40 hours upon completion of acclimatization. This allows for rapid and efficient CHO-K1 suspension acclimatization, and the acclimated cells have high viability and rapid proliferation. In addition, the acclimated CHO-K1 cells are monodispersed, uniform in size, and round and translucent. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The following are the state diagrams of the CHO-K1 acclimated cells in Example 1: (a) the state diagram of the cells after passage on the 18th day of acclimation; (b) the state diagram of the cells on the 21st day of acclimation, in which the cells are individually dispersed, uniform in size, round and translucent.

[0025] Figure 2 The following are the cell status diagrams of CHO-K1 acclimated cells in Comparative Example 4: (a) Cell status diagram after passage on the 30th day of acclimation; (b) Cell status diagram on the 33rd day of acclimation, where some cells are clumped, uneven in size, and not round.

[0026] Figure 3 The following are the cell status diagrams of CHO-K1 acclimated cells in Comparative Example 5: (a) Cell status diagram after passage on the 27th day of acclimation; (b) Cell status diagram on the 30th day of acclimation, where some cells are clumped, uneven in size, and not round.

[0027] Figure 4 The following are the cell status diagrams of CHO-K1 acclimated cells in Comparative Example 6: (a) Cell status diagram after passage on the 30th day of acclimation; (b) Cell status diagram on the 33rd day of acclimation, where some cells are clumped, uneven in size, and not round. DETAILED DESCRIPTION

[0028] The present application is further described below with reference to examples. It should be understood that the examples are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0029] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein may be used in experiments or practical applications, the materials and methods are described herein below. In the event of a conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific examples, which are not intended to limit the scope of this application.

[0030] In response to the problems existing in the prior art, the present application provides a composition for acclimating CHO-K1, which is characterized by comprising yeast hydrolysate and L-glutamine.

[0031] In the composition, the content of the yeast hydrolyzate is 0.6-2wt%, for example, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt%, 1.5wt%, 1.6wt%, 1.7wt%, 1.8wt%, 1.9wt%, 2wt%, and any values ​​therebetween.

[0032] In the composition, the content of L-glutamine is 0.04-0.25wt%, for example, it can be 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.1wt%, 0.11wt%, 0.12wt%, 0.13wt%, 0.14wt%, 0.15wt%, 0.16wt%, 0.17wt%, 0.18wt%, 0.19wt%, 0.20wt%, 0.21wt%, 0.22wt%, 0.23wt%, 0.24wt%, 0.25wt%, and any value in between these values.

[0033] Furthermore, the composition further comprises a basal serum-free medium, wherein the basal serum-free medium is known in the art and can include various types of basal serum-free medium available in the art, such as HyperCHO growth medium, Maibang growth medium, etc.

[0034] In some embodiments, the yeast hydrolysate can be purchased commercially or prepared in-house. Commercially available products include, but are not limited to, Ultrafiltered Yeast Hydrolysate from FUJIFILM Irvine Scientific. Preparation in-house is a commonly used method for yeast hydrolysis and is not particularly limited as long as it ensures the yeast hydrolysate has conventional efficacy. For example, products obtained from pure yeast cells through enzymatic or chemical hydrolysis primarily contain amino acids, peptides, nucleotides, vitamins, and trace elements. Furthermore, the product can be obtained by hydrolyzing yeast cells catalyzed by autolytic enzymes or exogenous enzymes.

[0035] In some embodiments, the content of the basal serum-free medium is 80-99.36 wt%, preferably 90-99.36 wt%, and most preferably 97.75-99.36 wt%. For example, it can be 80wt%, 82wt%, 84wt%, 86wt%, 88wt%, 90wt%, 91wt%, 92wt%, 93wt%, 94wt%, 95wt%, 96wt%, 97wt%, 97.5wt%, 97.85wt%, 97.95wt%, 98.05wt%, 98.15wt%, 98.25wt%, 98.35wt%, 98.45wt%, 98.55wt%, 98.65wt%, 98.75wt%, 98.85wt%, 98.95wt%, 99.05wt%, 99.15wt%, 99.25wt%, 99.36wt%, and any value in between.

[0036] In some embodiments, the composition further includes excipients and / or bioactive substances. For example, the bioactive substances include plant-derived hydrolysates, microbial-derived hydrolysates, animal-derived hydrolysates, etc.; the excipients include carbon sources, nitrogen sources, vitamins, minerals, pH buffers, osmotic pressure regulators, etc.

[0037] In some embodiments, the composition for acclimating CHO-K1 comprises yeast hydrolysate, L-glutamine, and a basic serum-free medium; wherein the content of the yeast hydrolysate is 0.6-2 wt %, the content of L-glutamine is 0.04-0.25 wt %, and the content of the basic serum-free medium is 80-99.36 wt %.

[0038] In some embodiments, the composition for acclimating CHO-K1 comprises yeast hydrolysate, L-glutamine, and a basic serum-free medium; wherein the content of the yeast hydrolysate is 0.6-2 wt %, the content of L-glutamine is 0.04-0.25 wt %, and the content of the basic serum-free medium is 97.75-99.36 wt %.

[0039] In some embodiments, the composition for acclimating CHO-K1 consists of yeast hydrolysate, L-glutamine and basal serum-free medium; wherein the content of the yeast hydrolysate is 0.6-2 wt %, and the content of L-glutamine is 0.04-0.25 wt %.

[0040] The present application also provides a method for acclimating CHO-K1, comprising using any one of the above-mentioned compositions for acclimating CHO-K1.

[0041] In some embodiments, the acclimation method comprises the following steps:

[0042] 1. Cell recovery step: Subculture the revived CHO-K1 to restore the state of CHO-K1;

[0043] 2. Cell acclimation step: inoculating the recovered CHO-K1 into the above-mentioned composition for acclimating CHO-K1 for subculture to acclimate CHO-K1.

[0044] CHO-K1 cells can be revived by methods known in the art. For example, cryopreserved CHO-K1 cells can be removed from a low-temperature environment and restored to a normal growth state. Furthermore, cryopreserved CHO-K1 cells can be removed from liquid nitrogen or -80°C and rapidly revived to 37°C to regain activity.

[0045] The state of CHO-K1 cells can be restored by methods known in the art, for example, the revived CHO-K1 cells are passaged to return to normal growth, metabolism and function.

[0046] In some embodiments, the acclimation method comprises the following steps:

[0047] 1. Steps to restore cells:

[0048] Step 1: Use a medium containing FBS to culture the revived CHO-K1 cells. Culture continuously until the cells recover, and then continue culturing with a medium containing FBS.

[0049] Step 2: When the cell density to be cultured reaches ≥80%, the cells are first digested with protease, and then basic serum-free medium is added to resuspend and wash the cells.

[0050] 2. Steps for acclimating cells:

[0051] Step 3: Use the above-mentioned composition for acclimating CHO-K1 to acclimate the CHO-K1 in step 2 to obtain acclimated CHO-K1.

[0052] In some embodiments, the acclimation method comprises the following steps:

[0053] Step 1: Cultivate the revived CHO-K1 cells in Ham's F-12K (Merck) medium containing 10% FBS and then place them in a 37°C, 5% CO2 incubator. When the cell density reaches approximately 90% confluency, subculture the cells. After two consecutive generations of culture to allow the cells to recover, the cells are seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimatized for two generations.

[0054] Step 2: When the cell density reaches approximately 85% confluency, wash with PBS and add 2 mL of 0.25% trypsin to digest the cells. After cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300 g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend and wash the cells.

[0055] 2. Steps for acclimating cells:

[0056] Step 3: Use the above-mentioned composition for acclimating CHO-K1 to acclimate the CHO-K1 in step 2 to obtain acclimated CHO-K1.

[0057] In some embodiments, the recovered CHO-K1 obtained in step 2 is added at a concentration of 0.3 to 0.5×10 6 cells / mL were inoculated into the composition for acclimating CHO-K1.

[0058] Using the acclimation method of the present application, the total time required to achieve a CHO-K1 cell viability of 95% or greater for three consecutive generations is 25 days or less, and the CHO-K1 cell doubling time is 40 hours or less upon completion of acclimation. Furthermore, upon completion of acclimation, the CHO-K1 cells are individually dispersed, uniform in size, and round and translucent.

[0059] The present application also provides the use of the above composition in acclimating CHO-K1.

[0060] The composition of the present application uses a yeast hydrolyzate and L-glutamine of a specific composition. When used to acclimate CHO-K1, the total time required for the CHO-K1 cell viability to be greater than or equal to 95% for three consecutive generations is less than or equal to 25 days. Upon completion of acclimatization, the CHO-K1 cell doubling time is less than or equal to 40 hours, thereby achieving rapid and efficient CHO-K1 suspension acclimatization. Furthermore, the acclimated CHO-K1 cells have a high viability and a rapid proliferation rate. Furthermore, the acclimated CHO-K1 cells are monodispersed, uniform in size, and round and translucent.

[0061] Example

[0062] The HyperCHO growth medium used in the following examples and comparative examples was purchased from Bloomage Biotech (Hainan) Co., Ltd. with the product number 80206; the Maibang growth medium was purchased from Shanghai Maibang Biotechnology Co., Ltd. with the product number MB1114.102.

[0063] Yeast hydrolysate was purchased from FUJIFILM Irvine Scientific with a product number of 96863 and a product name of Ultrafiltered Yeast Hydrolysate.

[0064] CHO-K1 adherent cells were purchased from ATCC with the catalog number CCL-61.

[0065] Ham's F-12K basal medium containing 10% fetal bovine serum was purchased from Merck (catalog number: N3520). Ham's F-12K basal medium containing 5% fetal bovine serum was purchased from Merck (catalog number: N3520).

[0066] PBS is the most widely used buffer in biochemical research. Its main components are Na2HPO4, KH2PO4, NaCl and KCl. It is generally used as a solvent to dissolve protective reagents.

[0067] Example 1

[0068] 1. Steps to restore cell status

[0069] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded into a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, the cells were subcultured. After two consecutive generations of culture to allow the cells to recover, the cells were seeded into Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0070] Step 2: When the cell density cultured in step 1 reaches about 85% fusion, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0071] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0072] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6 Cells were inoculated into HyperCHO acclimation medium (98.9124 wt% HyperCHO growth medium, 1 wt% yeast hydrolysate, and 0.0876 wt% L-glutamine) at a concentration of 10 cells / mL. Acclimation was successful until cell viability was ≥95% for three consecutive generations, and the cells were individually dispersed, uniform in size, round, and translucent. The total time from when cell viability was ≥95% for three consecutive generations was 15 days, and successful acclimation was achieved with a cell doubling time of approximately 29.5 hours.

[0073] After 36 days of continuous culture, the results showed that on the 15th to 36th day, the cell viability of each generation was higher than 95%, and the cells were individually dispersed, uniform in size, round and translucent CHO-K1, and adapted to the suspension cells in chemically defined culture medium ( Figure 1 ).

[0074] Example 2

[0075] 1. Steps to restore cell status

[0076] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded in a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, the cells were subcultured. After two generations of continuous culture to allow the cells to recover, the cells were seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0077] Step 2: When the cell density cultured in step 1 reaches about 85% fusion, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0078] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0079] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6 Cells were inoculated into HyperCHO acclimation medium (containing 99.3124 wt% HyperCHO growth medium, 0.6 wt% yeast hydrolysate, and 0.0876 wt% L-glutamine) and cultured until cell viability was ≥95% for three consecutive generations and the cells were individually dispersed, uniform in size, round, and translucent, indicating successful cell acclimation. The total time from when cell viability was ≥95% for three consecutive generations was 18 days, and the cell doubling time for successful acclimation was 29.8 hours.

[0080] After 36 days of continuous culture, the results showed that on days 18-36, the cell viability of each generation was higher than 95%, and the cells were individually dispersed, uniform in size, round and translucent CHO-K1, and were suspension cells adapted to chemically defined culture medium.

[0081] Example 3

[0082] 1. Steps to restore cell status

[0083] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded in a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, they were subcultured. After two generations of continuous culture to allow the cells to recover, the cells were seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0084] Step 2: When the cell density cultured in step 1 reaches approximately 85% confluency, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300 g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0085] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0086] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6 Cells were inoculated into HyperCHO acclimation medium (98.4124 wt% HyperCHO growth medium, 1.5 wt% yeast hydrolysate, and 0.0876 wt% L-glutamine) and cultured until cell viability was ≥95% for three consecutive generations and the cells were individually dispersed, uniform in size, round, and translucent, indicating successful cell acclimation. The total time required for achieving a cell viability of ≥95% for three consecutive generations was 18 days, and the cell doubling time for successful acclimation was 28.5 hours.

[0087] After 36 days of continuous culture, the results showed that on days 18-36, the cell viability of each generation was higher than 95%, and the cells were individually dispersed, uniform in size, round and translucent CHO-K1, and were suspension cells adapted to chemically defined culture medium.

[0088] Example 4

[0089] 1. Steps to restore cell status

[0090] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded in a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, the cells were subcultured. After two generations of continuous culture to allow the cells to recover, the cells were seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0091] Step 2: When the cell density cultured in step 1 reaches about 85% fusion, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0092] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0093] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6Cells were inoculated into HyperCHO acclimation medium (97.9124 wt% HyperCHO growth medium, 2 wt% yeast hydrolysate, and 0.0876 wt% L-glutamine) at 400 μg / mL. Acclimation was successful when cell viability was ≥95% for three consecutive generations and the cells were individually dispersed, uniform in size, round, and translucent. The total time required for achieving a cell viability of ≥95% for three consecutive generations was 21 days, and the cell doubling time for successful acclimation was 29.7 hours.

[0094] After 36 days of continuous culture, the results showed that on days 21-36, the cell viability of each generation was higher than 95%, and the cells were individually dispersed, uniform in size, round and translucent CHO-K1, and were suspension cells adapted to chemically defined culture medium.

[0095] Example 5

[0096] 1. Steps to restore cell status

[0097] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded in a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, the cells were subcultured. After two generations of continuous culture to allow the cells to recover, the cells were seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0098] Step 2: When the cell density cultured in step 1 reaches about 85% fusion, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0099] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0100] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6Cells were inoculated into HyperCHO acclimation medium (98.9562 wt% HyperCHO growth medium, 1 wt% yeast hydrolysate, and 0.0438 wt% L-glutamine) and cultured until cell viability was ≥95% for three consecutive generations and the cells were individually dispersed, uniform in size, round, and translucent, indicating successful cell acclimation. The total time required for achieving a cell viability of ≥95% for three consecutive generations was 21 days, and the cell doubling time for successful acclimation was 29.3 hours.

[0101] After 36 days of continuous culture, the results showed that on days 21-36, the cell viability of each generation was higher than 95%, and the cells were individually dispersed, uniform in size, round and translucent CHO-K1, and were suspension cells adapted to chemically defined culture medium.

[0102] Example 6

[0103] 1. Steps to restore cell status

[0104] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded in a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, the cells were subcultured. After two generations of continuous culture to allow the cells to recover, the cells were seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0105] Step 2: When the cell density cultured in step 1 reaches about 85% fusion, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0106] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0107] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6Cells were inoculated into HyperCHO acclimation medium (98.8685 wt% HyperCHO growth medium, 1 wt% yeast hydrolysate, and 0.1315 wt% L-glutamine) and cultured until cell viability was ≥95% for three consecutive generations and the cells were individually dispersed, uniform in size, round, and translucent, indicating successful cell acclimation. The total time required for achieving a cell viability of ≥95% for three consecutive generations was 21 days, and the cell doubling time for successful acclimation was 31.6 hours.

[0108] After 36 days of continuous culture, the results showed that on days 21-36, the cell viability of each generation was higher than 95%, and the cells were individually dispersed, uniform in size, round and translucent CHO-K1, and were suspension cells adapted to chemically defined culture medium.

[0109] Example 7

[0110] 1. Steps to restore cell status

[0111] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded in a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, the cells were subcultured. After two generations of continuous culture to allow the cells to recover, the cells were seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0112] Step 2: When the cell density cultured in step 1 reaches about 85% fusion, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0113] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0114] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6 Cells were inoculated into HyperCHO acclimation medium (98.9124 wt% MaiBang Growth Medium, 1 wt% yeast hydrolysate, 0.0876 wt% L-glutamine) and cultured until cell viability was ≥95% for three consecutive generations, and the cells were individually dispersed, uniform in size, round, and translucent, indicating successful cell acclimation. The total time from when cell viability reached ≥95% for three consecutive generations was 21 days, and the cell doubling time for successful acclimation was 32.8 hours.

[0115] After 36 days of continuous culture, the results showed that on days 21-36, the cell viability of each generation was higher than 95%, and the cells were individually dispersed, uniform in size, round and translucent CHO-K1, and were suspension cells adapted to chemically defined culture medium.

[0116] Example 8

[0117] 1. Steps to restore cell status

[0118] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded in a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, the cells were subcultured. After two generations of continuous culture to allow the cells to recover, the cells were seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0119] Step 2: When the cell density cultured in step 1 reaches about 85% fusion, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0120] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0121] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6 Cells were inoculated into HyperCHO acclimation medium (98.7808 wt% HyperCHO growth medium, 1 wt% yeast hydrolysate, and 0.2192 wt% L-glutamine) and cultured until cell viability was ≥95% for three consecutive generations and the cells were individually dispersed, uniform in size, round, and translucent, indicating successful cell acclimation. The total time from when cell viability reached ≥95% for three consecutive generations was 21 days, and the cell doubling time was 34 hours for successful acclimation.

[0122] After 36 days of continuous culture, the results showed that on days 21-36, the cell viability of each generation was higher than 95%, and the cells were individually dispersed, uniform in size, round and translucent CHO-K1, and were suspension cells adapted to chemically defined culture medium.

[0123] control group

[0124] 1. Steps to restore cell status

[0125] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded in a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, the cells were subcultured. After two generations of continuous culture to allow the cells to recover, the cells were seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0126] Step 2: When the cell density cultured in step 1 reaches about 85% fusion, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0127] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0128] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6 cells / mL, inoculated into HyperCHO acclimation medium (containing only HyperCHO growth medium), and cultured continuously.

[0129] The results showed that on the 6th day, the cell viability was less than 5%, and the cell density was less than 5×10 3 cells / mL, acclimation failed.

[0130] Comparative Example 1

[0131] 1. Steps to restore cell status

[0132] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded in a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, the cells were subcultured. After two generations of continuous culture to allow the cells to recover, the cells were seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0133] Step 2: When the cell density cultured in step 1 reaches about 85% fusion, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0134] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0135] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6 cells / mL, inoculated into HyperCHO acclimation medium (containing 99.9562 wt % HyperCHO growth medium, 0.0438 wt % L-glutamine), and cultured continuously.

[0136] The results showed that on days 24-30, the cell viability of each generation was less than 25%, and the cell density was less than 1×10 4 cells / mL, acclimation failed.

[0137] Comparative Example 2

[0138] 1. Steps to restore cell status

[0139] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded in a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, the cells were subcultured. After two generations of continuous culture to allow the cells to recover, the cells were seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0140] Step 2: When the cell density cultured in step 1 reaches about 85% fusion, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0141] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0142] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6cells / mL, inoculated into HyperCHO acclimation medium (containing 99.9124 wt% HyperCHO growth medium, 0.0876 wt% L-glutamine), and cultured continuously.

[0143] The results showed that on days 24-30, the cell viability of each generation was less than 45%, and the cell density was less than 1×10 4 cells / mL, acclimation failed.

[0144] Comparative Example 3

[0145] 1. Steps to restore cell status

[0146] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded in a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, the cells were subcultured. After two generations of continuous culture to allow the cells to recover, the cells were seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0147] Step 2: When the cell density cultured in step 1 reaches about 85% fusion, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0148] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0149] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6 cells / mL, inoculated into HyperCHO acclimation medium (containing 99.8685 wt% HyperCHO growth medium, 0.1315 wt% L-glutamine), and cultured continuously.

[0150] The results showed that on days 21-27, the cell viability of each generation was less than 45%, and the cell density was less than 1×10 4 cells / mL, acclimation failed.

[0151] Comparative Example 4

[0152] 1. Steps to restore cell status

[0153] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded in a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, the cells were subcultured. After two generations of continuous culture to allow the cells to recover, the cells were seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0154] Step 2: When the cell density cultured in step 1 reaches about 85% fusion, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0155] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0156] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6 cells / mL, inoculated into HyperCHO acclimation medium (containing 99.6 wt % HyperCHO growth medium, 0.4 wt % yeast hydrolysate), and cultured continuously.

[0157] The results showed that: on days 30-36, the cell viability of each generation was higher than 95%, and the doubling time was about 25.9h, but some CHO-K1 cells were clumped, uneven in size, and not round ( Figure 2 ).

[0158] Comparative Example 5

[0159] 1. Steps to restore cell status

[0160] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded in a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, the cells were subcultured. After two generations of continuous culture to allow the cells to recover, the cells were seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0161] Step 2: When the cell density cultured in step 1 reaches about 85% fusion, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0162] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0163] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6 cells / mL, inoculated into HyperCHO acclimation medium (containing 99 wt% HyperCHO growth medium, 1 wt% yeast hydrolysate), and cultured continuously.

[0164] The results showed that on days 30-36, the cell viability of each generation was higher than 95%, and the doubling time was about 25.4h, but some CHO-K1 cells were clumped, uneven in size, and not round ( Figure 3 ).

[0165] Comparative Example 6

[0166] 1. Steps to restore cell status

[0167] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded in a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, the cells were subcultured. After two generations of continuous culture to allow the cells to recover, the cells were seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0168] Step 2: When the cell density cultured in step 1 reaches about 85% fusion, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0169] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0170] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6cells / mL, inoculated into HyperCHO acclimation medium (containing 98 wt% HyperCHO growth medium, 2 wt% yeast hydrolysate), and cultured continuously.

[0171] The results showed that on days 30-36, the cell viability of each generation was higher than 95%, and the doubling time was about 38.8h, but some CHO-K1 cells were clumped, uneven in size, and not round ( Figure 4 ).

[0172] Comparative Example 7

[0173] 1. Steps to restore cell status

[0174] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded in a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, the cells were subcultured. After two generations of continuous culture to allow the cells to recover, the cells were seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0175] Step 2: When the cell density cultured in step 1 reaches about 85% fusion, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0176] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0177] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6 cells / mL, inoculated into HyperCHO acclimation medium (containing 89.9124 wt% HyperCHO growth medium, 10 wt% yeast hydrolysate, 0.0876 wt% L-glutamine), and cultured continuously.

[0178] The results showed that on days 21-27, the cell viability of each generation was less than 30%, the doubling time was greater than 100 h, and the cell density was less than 5×10 4 cells / mL, acclimation failed.

[0179] Comparative Example 8

[0180] 1. Steps to restore cell status

[0181] Step 1: CHO-K1 cells were revived and cultured in Ham's F-12K (Merck) medium containing 10% FBS. The cells were seeded in a T25 flask and then placed in a 37°C, 5% CO2 incubator. When the cell density reached approximately 90% confluency, the cells were subcultured. After two generations of continuous culture to allow the cells to recover, the cells were seeded in Ham's F-12K (Merck) medium containing 5% FBS and acclimated for two generations.

[0182] Step 2: When the cell density cultured in step 1 reaches about 85% fusion, discard the culture medium, wash twice with PBS, discard the PBS, add 2 mL of 0.25% trypsin to digest the cells, and after cell digestion is complete, add 4 mL of Ham's F-12K (Merck) medium containing 5% FBS to terminate digestion. After centrifugation at 300g for 5 minutes, discard the supernatant, add HyperCHO growth medium, and resuspend the washed cells.

[0183] 2. Steps for acclimating cells and verifying cell activity after acclimation

[0184] Step 3: Centrifuge the cells in step 2 at 300g for 5 minutes, discard the supernatant, and centrifuge at 0.3-0.5×10 6 Cells were inoculated into HyperCHO acclimation medium (99.5124 wt% HyperCHO growth medium, 0.4 wt% yeast hydrolysate, and 0.0876 wt% L-glutamine) and cultured until cell viability was ≥95% for three consecutive generations and the cells were individually dispersed, uniform in size, round, and translucent, indicating successful cell acclimation. The total time from when cell viability reached ≥95% for three consecutive generations was 27 days, and the cell doubling time for successful acclimation was 32.4 hours.

[0185] After 36 days of continuous culture, the results showed that on days 27-36, the cell viability of each generation was higher than 95%, and the cells were individually dispersed, uniform in size, round and translucent CHO-K1, and were suspension cells adapted to chemically defined culture medium.

[0186] The acclimation conditions and results of the above embodiments, control examples and comparative examples are shown in Table 1.

[0187] Table 1

[0188]

[0189]

[0190] The experimental data in Table 1 demonstrates that Examples 1-8 acclimate CHO-K1 cells in HyperCHO medium by adding yeast hydrolysate and L-glutamine. The results demonstrate that cell viability remains ≥95% for three consecutive generations, starting within 21 days. Furthermore, the CHO-K1 cells are individually dispersed, uniform in size, round, and translucent, and are well-adapted to suspension culture in chemically defined medium.

[0191] The control group, devoid of the active ingredients yeast hydrolysate and L-glutamine, was maintained in HyperCHO medium alone. Comparative Examples 1-3, however, added specific amounts of L-glutamine as the active ingredient and were acclimated in HyperCHO medium. Results showed that both the control group and Comparative Examples 1-3 failed due to low cell viability, preventing effective survival and thus failing to complete acclimation.

[0192] Comparative Examples 4-6 added specific amounts of active components from yeast hydrolysate and were acclimated in HyperCHO medium. Results showed that cell viability remained ≥95% for three consecutive generations, starting after more than 27 days. Furthermore, some cells clumped, exhibited uneven size, and were not round. Due to the prolonged acclimation period and the defects in cell morphology that did not meet the requirements, acclimation could not be completed.

[0193] Comparative Example 7: Excessive yeast hydrolyzate and L-glutamine were added and the cells were acclimated in HyperCHO medium. The results showed that the acclimation failed because the cell viability was too low and the cells could not survive effectively, and the acclimation could not be completed.

[0194] Comparative Example 8 employed insufficient amounts of yeast hydrolysate and L-glutamine for acclimation in HyperCHO medium. Results showed that cell viability remained ≥95% for three consecutive generations, starting on day 27. Furthermore, some cells clumped, exhibited uneven size, and were not round. Acclimation took a long time, and the cells did not meet the requirements. This suggests that the amount of yeast hydrolysate used affects acclimation results.

[0195] This demonstrates that the composition obtained from yeast hydrolyzate and L-glutamine in the specific ratio claimed in this application can provide a CHO-K1 acclimation pathway in serum-free medium with a short acclimation time, high success rate, and excellent efficacy, resulting in a single, dispersed, uniformly sized, round, and translucent suspension cell line with high viability. The composition obtained from yeast hydrolyzate and L-glutamine in the specific ratio exhibits a synergistic effect in the CHO-K1 acclimation process.

[0196] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. A composition for acclimating CHO-K1, characterized in that The invention comprises yeast hydrolyzate and L-glutamine; wherein the content of the yeast hydrolyzate is 0.6-2wt%, and the content of the L-glutamine is 0.04-0.25wt%. 2 . The composition for acclimating CHO-K1 according to claim 1 , further comprising a basal serum-free medium. 3 . The composition for acclimating CHO-K1 according to claim 2 , wherein the basal serum-free medium is one or a combination of two of HyperCHO growth medium and Maibang growth medium.

4. The composition for acclimating CHO-K1 according to any one of claims 1 to 3, characterized in that The acclimated CHO-K1 cells were individually dispersed, uniform in size, round and translucent. A method for acclimating CHO-K1, comprising acclimating CHO-K1 using the composition for acclimating CHO-K1 according to any one of claims 1 to 4.

6. The acclimation method according to claim 5, wherein the acclimation method comprises the following steps: 1) Subculturing the revived CHO-K1 cells to restore the state of CHO-K1; 2) The recovered CHO-K1 cells are inoculated into the composition for acclimating CHO-K1 and subcultured to acclimate CHO-K1.

7. The acclimation method according to claim 6, wherein the recovered CHO-K1 is heated at 0.3 to 0.5 × 10 6 cells / mL were inoculated into the composition for acclimating CHO-K1.

8. The acclimation method according to claim 6, wherein the total time from the start of the CHO-K1 cell viability being greater than or equal to 95% for three consecutive generations is less than or equal to 25 days, and the doubling time of CHO-K1 is less than or equal to 40 hours when acclimation is completed.

9. The acclimation method according to claim 6, wherein the acclimated CHO-K1 cells are individually dispersed, uniform in size, round and translucent.

10. Use of the composition according to any one of claims 1 to 4 in acclimating CHO-K1.

Images