CN111748516B

CN111748516B - Method for preparing suspension cells by rapid optimization of agglomerated cell suspension

Info

Publication number: CN111748516B
Application number: CN201910250806.9A
Authority: CN
Inventors: 陈平; 祝志刚
Original assignee: Jiaxing Anyu Biotechnology Co ltd
Current assignee: Jiaxing Anyu Biotechnology Co ltd
Priority date: 2019-03-29
Filing date: 2019-03-29
Publication date: 2024-04-12
Anticipated expiration: 2039-03-29
Also published as: CN111748516A

Abstract

The invention belongs to the technical field of cell biology, and discloses a method for preparing suspension cells by rapidly optimizing an agglomerated cell suspension. The invention adopts Ackutase enzyme to digest the agglomerated cells, uses a serum-free culture medium to neutralize the digestive enzyme after the digestion is finished, and then uses the serum-free culture medium to resuspend the cells to obtain the suspension cells. On the one hand, the method can quickly and effectively suspend the agglomerated cells, shortens the time for domesticating the suspended cells, and also keeps the activity of the cells to the greatest extent; on the other hand, the serum-free culture medium is adopted, the components of the culture medium are definite, no exogenous serum component is contained, the proliferation capacity and stability of cells are obviously improved, and the method has good application prospect.

Description

Method for preparing suspension cells by rapid optimization of agglomerated cell suspension

Technical Field

The invention belongs to the technical field of cell biology, and particularly relates to a method for preparing suspension cells by rapidly optimizing an agglomerated cell suspension.

Background

The large-scale mammalian cell culturing technology is an effective method for producing recombinant protein, virus vaccine and other biological products in biological pharmaceutical enterprises, and the traditional cell culture in China adopts an adherent cell culture mode. The method is simple and convenient to operate, the cell density is low due to the limited surface area of the rotary bottle, the product yield is low, serum is required to be added during culture, animal viruses or mycoplasma which are not fully inactivated can be carried due to the fact that the chemical composition of the serum is uncertain, and the use of the serum not only increases the cost input, but also increases the instability of the product and the difficulty of downstream purification. Therefore, the serum-free suspension culture of the mammalian cells is realized, and the method has important significance for improving the productivity, reducing the cost and simplifying the downstream production.

The general way of adherent cell suspension acclimation is to gradually reduce the serum content of the culture medium to gradually adapt the cells to the serum-free suspension culture medium, so that by acclimation, various mammalian cells are changed from an adherent state into a cell line which can be grown in suspension in the serum-free culture medium, and more cells can be grown in unit volume. However, the acclimatized suspension cells are cultured for a period of time to cause aggregation of cells in the culture solutionFor example, the phenomenon of aggregation may occur under the condition of culturing by replacing the other medium. The occurrence of cell clumping is due to several factors: (1) In the process of culturing cells, the cell viability is reduced, the cell death rate is increased, and DNA released into the suspension after the cells are ruptured can cause the cells in the suspension to gradually agglomerate; (2) Ca in serum-free Medium ²⁺ The ion concentration, the dextran sodium sulfate concentration, the heparin sodium concentration and other components can have certain influence on cell aggregation, especially the dextran sodium sulfate and the heparin sodium play roles in the culture medium in resisting cell aggregation, when the cell density in the nutrient solution is increased to a certain density along with the increase of the cell density in the culture medium (for example, the HEK293 cell density reaches 3-5 multiplied by 10) ⁶ cells/ml), the anti-cell aggregation effect of the anti-cell aggregation component in the nutrient solution on cells is reduced, so that the mutually aggregated cells are gradually increased; (3) Suspension cells are not completely acclimatized to suspension, resulting in cell clumping during culture. For the problem of re-agglomeration of suspended cells during the culture, there are generally two methods adopted, the first method is to re-suspend and digest cells using pancreatin, terminate the pancreatin digestion with 2% fbs (DMEN), and then re-suspend the cells with serum-free cell nutrient solution; the second method is that the heavy head starts to domesticate, the pancreatin digestion is adopted, the serum content of the culture medium is gradually reduced, from 10% FBS to 1% FBS, when the cells adapt to the culture medium containing 1% FBS for several generations, the DMEN and the serum-free culture medium gradually adapt to the complete serum-free culture medium according to 95% -90% -75% -50% -25% -10% -5% -0%. In both methods, pancreatin is used for re-digestion, and agglomerated cells are re-domesticated to become suspension cells through repeated liquid exchange and frequent seed separation. However, the cells which are again agglomerated after being acclimatized to a suspension state are very fragile and sensitive to the external environment, and after the cells are agglomerated, the cells are prepared to be in a single suspension cell culture state by using a traditional method again, wherein the acclimation time is as long as one month, the cells are easily damaged by digestive enzymes to reduce the activity, and the activity of pancreatin is stopped by using fetal bovine serum, so that serum components are inevitably introduced in the cell culture process.

Disclosure of Invention

The invention aims at solving the technical problems that after the cells subjected to suspension domestication are aggregated again, the aggregated cells are digested, so that the cells become single free suspension cells again, the prepared suspension cells can survive and grow healthily in a serum-free culture medium, and the phenomenon of cell aggregation does not occur in subsequent amplification culture.

In view of the above, the present invention provides a method for the rapid and optimal preparation of suspension cells from a suspension of agglomerated cells.

Preferably, the method comprises the steps of: the agglomerated cells are digested by Accutase enzyme, the digestive enzyme is neutralized after the digestion is finished, and then the cells are resuspended by serum-free medium to obtain suspended cells.

Preferably, the step of diluting the Actuase enzyme is further included before the step of digesting the pelleted cells with the Actuase enzyme.

Preferably, ca-free materials are used ²⁺ And Mg (magnesium) ²⁺ More than 5 times diluted Ackutase enzyme.

Preferably, the neutralizing digestive enzyme uses more than 5 times of the volume of the serum-free medium.

Preferably, ca of the serum-free medium ²⁺ And Mg (magnesium) ²⁺ The content is 0.05 mmol/L-0.2 mmol/L.

Preferably, the digestion temperature is 35-38 ℃ and the digestion time is 2-3min.

Preferably, the method further comprises the steps of shaking the agglomerated cell suspension by a shaker and filtering the agglomerated cell suspension before the step of digesting the agglomerated cells by using an Ackutase enzyme, wherein a filter screen with a diameter of 40 μm is adopted for the filtering step.

Preferably, the filter screen is a 40 μm cell screen.

In the invention, a cell sieve sterilized by 40 mu m is used for screening cells in suspension culture during the subculture, large-particle cell clusters are filtered, and only cell clusters with uniform size are left, so that a suspension cell line with uniform size and good growth condition can be established by the method.

The method of the invention is applicable to a variety of mammalian cells, such as HEK293 cells, a549 cells, PK15 cells, etc.

A second object of the present invention is to provide the use of a method for preparing suspension cells for suspension culture of mammalian cells.

Preferably, the method for preparing suspension cells comprises the following steps: the agglomerated cells are digested by Accutase enzyme, the digestive enzyme is neutralized after the digestion is finished, and then the cells are resuspended by serum-free medium to obtain suspended cells.

Preferably, the filter screen is a 40 μm cell screen.

Preferably, the mammalian cells are any one of HEK293 cells, a549 cells and PK15 cells.

The method can effectively solve the problem that the suspension cells are aggregated or clustered again in the culture process, so that the clustered cells can be rapidly optimized into a suspension state, thereby improving the in-vitro proliferation level of the cells.

A third object of the present invention is to provide the use of a method for preparing suspension cells for the production of recombinant proteins or viral vaccines.

By optimizing the cell culture process, continuous and stable suspension culture of cells in a serum-free culture medium is realized, so that the in-vitro proliferation level of the cells is improved, and the productivity of biological products such as recombinant proteins or virus vaccines is improved.

The beneficial effects are that:

(1) Aiming at the problem of cell sensitivity, the invention uses Accutase enzyme for digestion, the concentration of the Accutase enzyme used is low, the activity is high, the effect is mild, the cell membrane and the surface epitope of the cell cannot be damaged, and the Accutase does not contain any protein derived from mammals or bacteria, BSE, parvovirus and other common viruses in trypsin, and has high safety.

(2) The method aims at the fact that the cells in suspension culture are acclimatized again after being clustered in the culture process, and compared with the traditional re-acclimation mode, the method does not need to take a long time, only needs 1 day for cell acclimation to single suspension cells, and has the characteristics of being simple in operation and quick in acclimation.

(3) The invention uses the serum-free culture medium to stop digestion, effectively eliminates the defects of pathogens, adsorption factors and the like in the serum of animal sources, and simultaneously, the serum-free culture medium contains various growth factors and nutrient elements, thereby effectively improving the proliferation capability of cells.

(4) The suspension cells prepared by the method have stable single state, no cell agglomeration phenomenon can occur in continuous culture for multiple generations, and the stability is high.

Drawings

FIG. 1 shows that HEK293 suspension culture density was observed to be 3X 10 before acclimation in example 1 ⁶ Severe cell clumping was seen at 100 x magnification at each/mL.

FIG. 2 shows the result of observation under a microscope of a cell suspension after passing through a 40 μm cell sieve after shaking of a shaker, at a magnification of 100 times.

FIG. 3 shows that the cell culture density of 5X 10 is observed by diluting the cell suspension 4-fold after acclimation by the method of the present invention in example 1 ⁶ Cell clumping was not seen in each mL, magnification 100-fold.

FIG. 4 is a graphical representation of the small amount of cell clumping observed after cell culture for 25 passages at 100 x magnification of 4 x dilution of cell suspension acclimated using the method of the invention of example 1.

Detailed Description

The present invention will be described in more detail with reference to examples. It should be understood that the practice of the invention is not limited to the following examples, but is intended to be within the scope of the invention in any form and/or modification thereof.

In the present invention, unless otherwise specified, all dilution factors are volume units, and all equipment, raw materials, etc. are commercially available or are commonly used in the industry. The methods employed in the examples are those generally known in the art, unless otherwise indicated.

In the present invention, the AccUtase enzyme used was purchased from gibco and Promocell, the original concentration of AccUtase enzyme was not disclosed; in the present invention, the serum-free medium used is a serum-free medium commonly used in the art for cell culture, and any serum-free medium that can be used for cell culture is suitable for the present invention, ca requiring a serum-free medium ²⁺ And Mg (magnesium) ²⁺ The content is 0.05 mmol/L-0.2 mmol/L.

Example 1

A method for preparing HEK293 suspension cells by rapid optimization of agglomerated cells comprises the following steps:

optimization of Accutase enzyme digestion concentration, digestion time and termination of digestion

The concentration of AccUtase enzyme required for different cells is different, and in the case of unknown primary enzyme concentration, the appropriate concentration of AccUtase enzyme capable of digesting the agglomerated cells needs to be selected.

Reagent: ackutase enzyme (available from gibco and Promocell) PBS buffer (pH 7.4) without Ca2+ and Mg2+, serum-free medium (available from gibco, ca) ²⁺ And Mg (magnesium) ²⁺ HEK293 cell suspension with the content of 0.05mmol/L to 0.2 mmol/L).

1. Sampling and counting from a bottle of the aggregated cell suspension, taking 2 plates of 12-well plates, and the cell density of each well is 1 multiplied by 10 ⁶ Shaking 1mL of cell suspension per well, and placing into a carbon dioxide incubator at 37deg.CAnd (5) standing and culturing for one day. The clustered cell suspension is HEK293 cells which are subjected to suspension culture after domestication, and when the culture density reaches 3×10 ⁶ At each mL, the cell suspension showed significant agglomeration, as shown in FIG. 1.

2. Ackutase enzyme is diluted by PBS buffer solution for 2 times, 5 times, 7 times, 10 times and 15 times respectively, and Ackutase enzyme with different dilution times is obtained.

3. A plate of 12-well plate was taken out from the carbon dioxide incubator, cells were digested with different concentrations of acctase enzyme, each concentration digested with 2 wells, the amount of acctase enzyme added was 250 μl/well, the degree of digestion of cells per well and the individual state of the digested cells were observed under an optical microscope during digestion, and the number of cells and the cell viability were counted by a hemocytometer or CountStar cell counter, and the experimental results are shown in table 1 below. It can be seen that the effect of the AccUtase enzyme diluted by more than 5 times is better than that of the AccUtase enzyme diluted by 2 times, wherein the cell activity is higher and the digestion time is short after the AccUtase enzyme diluted by 5 times is adopted for digestion, so that the AccUtase enzyme diluted by 5 times is preferably adopted for digestion, and the digestion time is preferably 2-3min.

TABLE 1 digestion effects of Ackutase enzymes at different dilution factors

It should be understood that dilution of Ackutase enzyme is preferably performed without Ca ²⁺ 、Mg ²⁺ Ultrapure water and any other buffer solution containing no Ca can be used ²⁺ 、Mg ²⁺ Or a buffer solution. Ca (Ca) ²⁺ And Mg (magnesium) ²⁺ The concentration has a significant effect on cell clumping, and the inventors found that Ca when in serum-free medium ²⁺ And Mg (magnesium) ²⁺ At a concentration higher than 0.2mmol/L, the cells have a marked tendency to agglomerate, indicating a high concentration of Ca ²⁺ And Mg (magnesium) ²⁺ Is beneficial to cell adherence culture and is not beneficial to cell suspension culture, therefore, in order to avoid Ca ²⁺ And Mg (magnesium) ²⁺ Has an influence on the digestion effect of Ackutase, and preferably no Ca is used ²⁺ 、Mg ²⁺ The Ackutase enzyme was diluted.

4. The Accutase enzyme diluted 5 times by PBS buffer is diluted 1, 2, 3, 4, 5, 7, 8, 9, 10, 11 and 12 times by serum-free culture medium respectively to obtain Accutase enzyme solutions with different concentrations after dilution by serum-free culture medium, and the digestion effect of the Accutase enzyme diluted different times by serum-free culture medium on the agglomerated cells is observed, so that the neutralization of the digestive enzyme by the serum-free culture medium is determined.

5. Another plate, a 12-well plate, was removed from the carbon dioxide incubator, and different concentrations of Actutase enzyme diluted with serum-free medium were added, each concentration was digested to 1 well, the amount of Actutase enzyme added was 250. Mu.L/well, and the cell state was observed by an optical microscope, and the experimental results are shown in Table 2 below. It was found that the Accutase enzyme was unable to digest cells at 5-fold or more dilution with serum-free medium, indicating that the dilution of Accutase enzyme was able to completely terminate the cell digestion, i.e., neutralize the digestion of the digestive enzyme, at 5-fold or more dilution with serum-free medium.

TABLE 2 digestion effects of Ackutase enzyme at various fold dilutions with serum-free Medium

Serum-free medium:

the serum-free culture medium is a culture medium without adding animal-derived serum, and consists of a basic culture medium and additive factors such as hormone, growth factors, adherence factors, binding proteins, trace elements and the like in proper proportion. Common hormones such as glucagon, growth hormone, insulin, thyroxine, estradiol, hydrocortisone, progesterone and the like, common growth factors include polypeptide growth factors and steroid growth factors such as epidermal growth factor, fibroblast growth factor, nerve growth factor and the like, attachment factors include fibronectin, collagen, laminin, polylysine and the like, binding proteins include transferrin and albumin, trace elements such as selenium and the like. The basal medium is usually a synthetic medium comprising glucose, amino acids, inorganic salts, vitamins, etc. in a certain ratio, and is essential for maintaining the growth and metabolism of tissues or cells. The addition amount of certain components of the culture medium can be correspondingly adjusted when different cell strains are cultured, so that the nutrition requirements of the cell strains are better met, and the growth vigor of the cell strains is good.

Magnesium sulfate and calcium chloride are commonly used inorganic salts in basal media, and the inventors have found that by employing a medium containing Ca at various concentrations ²⁺ And Mg (magnesium) ²⁺ When 293 cells were cultured in suspension in a serum-free medium, ca was found in the serum-free medium ²⁺ And Mg (magnesium) ²⁺ At a concentration higher than 0.2mmol/L, the apparent tendency of cells to form clusters was observed under an optical microscope, indicating a high concentration of Ca ²⁺ And Mg (magnesium) ²⁺ Is favorable for cell adherence culture, and is unfavorable for cell suspension culture; while Ca in serum-free medium ²⁺ And Mg (magnesium) ²⁺ When the concentration of (C) is too low (less than 0.05 mmol/L), the proliferation capacity of the cells is found to be decreased by counting with a hemocytometer or a CountStar cytometer. Therefore, to avoid Ca ²⁺ And Mg (magnesium) ²⁺ Influencing cell suspension culture, improving cell culture efficiency, preferably Ca without serum culture medium ²⁺ And Mg (magnesium) ²⁺ The content is 0.05 mmol/L-0.2 mmol/L.

(II) preparation of suspension cells by rapid optimization

Reagent: the Ackutase enzyme was diluted 5-fold in PBS buffer, serum-free medium, HEK293 cell suspension.

1. 50mL of the cell suspension was pipetted into a centrifuge tube (labeled tube A) from a bottle of the cell suspension in which cell clumping had occurred, the lid was closed, and the tube was placed in a constant temperature shaker at 37℃and 200rpm and shaken for 10min. The cell suspension with the cell aggregation is HEK293 cells subjected to suspension culture after domestication, and when the culture density reaches 3×10 ⁶ At each mL, the cell suspension appeared to have significantly agglomerated, with a plurality of cell clusters containing a large number of cell counts, as shown in FIG. 1.

2. Another centrifuge tube (labeled tube B) was taken, tube B was opened in the safety cabinet, 1 40 μm cell sieve was placed on tube B, the supernatant in tube a was pipetted in the biosafety cabinet slowly into tube B, after all supernatant was added to tube B, the cell sieve was removed and tube B was capped, and centrifuged at 800rpm for 10min.

3. The supernatant from tube B was discarded in a biosafety cabinet, and 500. Mu.L of a lower cell suspension was left in tube B, and the cell mass was observed by electron microscopy, as shown in FIG. 2, and a large amount of cell mass remained in the cell suspension. To the cell suspension, PBS buffer was added to dilute 1.5mL of Ackutase enzyme 5-fold, and after gentle pipetting, tube B was placed in 37℃and rocked up for 2-3min at 150 rpm.

For larger cell clusters, cells in the center of the cluster undergo massive apoptosis or death, and cells in different layers can be rapidly separated by utilizing the different weights of the cell clusters with different cell numbers and combining the centrifugal force of a shaking table. After shaking the shaking table, the agglomerate in the test tube gradually becomes larger from top to bottom, the cell suspension with smaller cell agglomerate in the supernatant is taken out for sieving, so that cells with uniform agglomerate size can be screened out, dead cells in large agglomerate can be excluded, the aim of better controlling the digestion time during digestion is achieved, and cells with consistent effect of digested cells and higher cell activity are obtained.

4. The test tube B was removed, and 10mL of serum-free medium was added to the test tube B to stop the Actutase from digesting the cells, and after mixing, the cells were centrifuged at 800rpm for 10min. The purpose of centrifugation here was to remove the Ackutase enzyme, and the inventors have found by comparing the effects of centrifugation and non-centrifugation that the same effect of suspension cells can be obtained without centrifugation after neutralization of the digestive enzyme, indicating that the Ackutase enzyme after neutralization with serum-free medium does not affect the cell suspension culture.

5. The tube B was removed, the supernatant from tube B was discarded, and 10mL of serum-free medium was added to tube B to resuspend the cells. Sub-packaging the resuspended cells into centrifuge tubes, and counting the cell density per milliliter, with a cell density of 0.5X10 in each centrifuge tube ⁶ cells/mL, 10mL of cell suspension was contained in each centrifuge tube, and the centrifuge tubes were placed in a 37℃carbon dioxide incubator and cultured with shaking at 200 rpm.

6. When the cell density in each centrifuge tube reaches 3 to 5 multiplied by 10 ⁶ cell clumping did not occur at cells/mL, indicating successful cell acclimation to suspension cells that were stably passaged (FIG. 3).

Example 2

The stability of HEK293 suspension cells obtained in example 1 in serum-free medium was evaluated.

HEK293 cells obtained after further acclimation by the method of the invention are subjected to suspension culture in a serum-free medium, and the count and the survival rate are counted by using a CountStar cell counter every 24 hours and are 0.5X10 every 3 days ⁶ The cell/mL density was passaged. Through 25-generation growth stability evaluation, the growth of cells is found to be stable, only a small amount of cell agglomeration phenomenon occurs, which indicates that HEK293 cells which are domesticated into a suspension state again by adopting the method disclosed by the invention adapt to the nutrition environment of a serum-free culture medium, can be stably subjected to suspension culture, and grow well (figure 4).

The inventor adopts the same method to rapidly optimize the A549 cells which are subjected to suspension culture and have cell aggregation, and obtains the same experimental effect. The method is suitable for suspension culture of various mammalian cells, can acclimate the clustered cells into a suspension state capable of stabilizing multi-generation culture in a short time, and does not damage the cell viability.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

1. A method for the rapid optimisation of a suspension of cells in mass, characterised in that in the method: firstly adopting Accutase enzyme to digest the agglomerated cells, stopping the digestive enzyme after the digestion is finished, and then re-suspending the cells by using a serum-free culture medium to obtain suspended cells; wherein, the method further comprises the step of diluting the Actuase enzyme before adopting the Actuase enzyme to digest the agglomerated cells, and Ca is not contained ²⁺ And Mg (magnesium) ²⁺ Diluting Ackutase enzyme more than 5 times; stopping digestive enzyme by adopting more than 5 times of serum-free culture medium; in the serum-free culture medium, ca ²⁺ And Mg (magnesium) ²⁺ The content is 0.05 mmol/L-0.2 mmol/L; the method further comprises the steps of shaking table shaking and filtering the agglomerated cell suspension before the agglomerated cells are digested by Accutase enzyme, wherein a filter screen with the diameter of 40 mu m is adopted for filtering; the agglomerated cell suspension is HEK293 cells subjected to suspension culture after domestication.

2. The method according to claim 1, wherein the digestion temperature is 35-38 ℃ and the digestion time is 2-3min.

3. The method of claim 1, wherein the filter mesh is a 40 μm cell sieve.