A safe, efficient and low-cost cleaning solution for suspended cells and its application

Abstract

The application discloses a safe, efficient and low-cost cleaning solution for suspended cells and application thereof, which comprises multiple kinds of glucose, amino acid supplements, vitamin supplements, anti-shear protective agents and sodium bicarbonate, is designed for the harvesting and washing process of suspended cells, can effectively remove residual culture medium, cell fragments, metabolic waste and process-related impurities while effectively maintaining cell viability, viable cell density and normal morphology, so as to realize the harvesting target of high-purity, high-activity and low-risk material of suspended cells, and is especially suitable for the production process of high-demand cell products such as cell culture meat. The cleaning solution system can be adapted to the continuous harvesting process of a large-scale bioreactor, can be integrated with automatic harvesting equipment, realizes stable, efficient and repeatable cell collection and washing operation, and meets the full-scene requirements from laboratory research and development to industrial production.

Description

Technical Field

[0001] This invention belongs to the field of cell culture meat production technology, specifically relating to a safe, efficient, and low-cost cleaning solution for suspended cells and its application. Background Technology

[0002] As an emerging alternative protein production technology, cell-cultured meat technology has many advantages, such as being low-carbon and environmentally friendly, reducing the spread of pathogens, reducing the use of antibiotics, and avoiding animal welfare issues, providing a new approach to meat production.

[0003] Cultured meat is a type of edible meat product created by directly culturing animal-derived cells in a controlled artificial environment using biomanufacturing technology. Its core principle is to precisely mimic the natural growth patterns of muscle tissue within an animal, producing a product highly similar to natural meat in appearance, taste, and nutritional characteristics without relying on traditional livestock farming.

[0004] In the production of cultured meat, after large-scale expansion, suspended cells need to undergo a washing and harvesting process to remove impurities such as culture medium components, dead cells, cell debris, and metabolic waste to obtain purified cells that meet application standards. Traditional processes often use physiological saline as the washing solution. However, physiological saline cannot maintain cell viability and density during the washing process, leading to significant cell breakage and incomplete impurity removal, ultimately negatively impacting the quality stability of the cell product. Therefore, developing a novel, safe, efficient, and low-cost suspension cell washing solution formulation is of great significance for promoting the industrial application of cultured meat products.

[0005] This cleaning fluid is compatible with large-scale bioreactor continuous harvesting processes and can be integrated with automated harvesting equipment to achieve stable, efficient, and repeatable cell collection and cleaning operations, meeting the needs of all scenarios from laboratory research and development to industrial production. Summary of the Invention

[0006] To address the problems of significant cell viability loss and incomplete impurity removal in existing suspended cell washing processes, this invention provides a safe, efficient, and low-cost suspended cell washing solution. This solution is designed with reference to the composition of suspended cell culture media, achieving highly efficient washing while being safe and low-cost, thus effectively replacing the washing solutions used in traditional processes.

[0007] The technical solution of this invention is as follows: The first objective of this invention is to provide a safe, efficient, and low-cost washing solution for suspended cells, wherein the washing solution for suspended cells comprises multiple substances selected from glucose, amino acid supplements, vitamin supplements, shear protectants, and sodium bicarbonate. The amino acid supplement includes arginine hydrochloride, cystine, histidine hydrochloride monohydrate, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine. The vitamin supplement includes D-biotin, choline chloride, folic acid, inositol, nicotinamide, D-pantothenic acid•½Ca, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, and sodium chloride; The shear protectant is polyvinyl alcohol and xanthan gum; Furthermore, the total concentration of various substances added to the washing solution for suspending cells ranges from 9.65156 to 72.3867 mg / mL.

[0008] Furthermore, the total concentration of various substances added to the washing solution used for suspending cells ranges from 12.06445 to 60.32225 mg / mL.

[0009] Furthermore, the total concentration of various substances added to the washing solution used for suspending cells ranges from 24.1289 to 60.32225 mg / mL.

[0010] Furthermore, in the washing solution used for suspending cells, the concentration range of any added substance is 0.04~60 mg / mL.

[0011] Furthermore, in the washing solution used for suspending cells, the amount of each substance added is 0.4 to 3 times the following dosage reference: glucose 20 mg / mL, amino acid supplement 0.5584 mg / mL, vitamin supplement 0.4705 mg / mL, polyvinyl alcohol 1 mg / mL, sodium bicarbonate 2 mg / mL, and xanthan gum 0.1 mg / mL.

[0012] A second objective of this invention is to provide the application of the aforementioned cleaning solution for suspended cells in the cleaning of suspended cells, wherein the aforementioned cleaning solution is used to clean the suspended cells.

[0013] Furthermore, the aforementioned cleaning solution is used to clean the harvested suspended cells during scale-up production.

[0014] Furthermore, the washing solution for suspended cells can maintain cell viability, cell density, and cell morphology while washing.

[0015] Furthermore, the application includes the following steps: (1) Cell propagation: The YP-S4-S-SC cell line was scaled up step by step from shake flasks to reactors, and finally transferred to a 200 L bioreactor for scale-up culture. (2) Cell counting: When the cell density reaches 8×10⁶ cells / year, the cell count is determined by the cell count. 6 When the number of cells / mL is above a certain level, the harvested cells undergo continuous washing and harvesting. (3) Cell washing and harvesting: The amount of washing solution added in each round is 4 times the weight of the cell precipitate, and it is gently mixed. A butterfly centrifuge is used, and the centrifugation conditions are: centrifugal force 1000g / min, centrifugation temperature 2-8℃. The supernatant washing solution is discarded, the cell precipitate is retained, and the washing solution is added repeatedly until the washing is completed continuously.

[0016] Compared with existing cleaning solutions, the advantages of this application are as follows: (1) High cell activity: The cleaning solution can keep the survival rate of suspended cells stable at ≥90% after treatment; this indicator is significantly better than the survival rate of suspended cells after washing with physiological saline, highlighting the core advantage of the appropriate cleaning solution in ensuring cell activity.

[0017] (2) High safety: The cleaning solution has a simple formula and does not contain dangerous substances, so it will not cause subsequent cell safety issues.

[0018] (3) High efficiency: The cleaning solution can efficiently remove most of the culture medium residues, cell debris and small molecule impurities in the system. After sample testing, all residual substance indicators meet the quality requirements of cell culture meat for cell purification products.

[0019] (4) Low cost: The cleaning solution has a simple formula and does not contain any expensive substances, so the cost is low. Attached Figure Description

[0020] Figure 1 Statistical graphs of viable cell density at 0.5 h and 3 h after washing suspended cells with washing solutions containing different additives.

[0021] Figure 2 Statistical graphs of cell viability at 0.5 h and 3 h after washing suspended cells with washing solutions containing different additives.

[0022] Figure 3 Statistical graphs of cell viability and viable cell density at four time points (0.5, 1, 2, and 3 h) after washing cells with washing solution 1.

[0023] Figure 4 Bright-field photographs comparing cell morphology before and after washing with washing solution 1. A is the cell morphology before washing, and B is the cell morphology after washing with washing solution 1 for 3 hours.

[0024] Figure 5 Statistical graph showing cell viability and viable cell density at four time points (0.5, 1, 2, and 3 h) after washing cells with physiological saline.

[0025] Figure 6 Bright-field photographs comparing cell morphology before and after washing with physiological saline. A is the cell morphology before washing, and B is the cell morphology after washing with physiological saline for 3 hours.

[0026] Figure 7 The graph shows the trend of residue concentration over time during the cleaning process of biomass using cleaning solution 1. The sampling points were 0, 0.5, 1, 2 and 3 h during the cleaning process. A represents forsocrine, B represents acetylcysteine ​​and C represents ethanolamine.

[0027] Figure 8 Statistical graphs of viable cell density at 0.5 h and 3 h after washing suspended cells with different concentrations of washing solution.

[0028] Figure 9 Statistical graphs of cell viability at 0.5 h and 3 h after washing suspended cells with different concentrations of washing solution.

[0029] Figure 10 In scale-up production, after cells have been continuously washed with the cleaning solution for four rounds, the statistical chart shows the cell viability and viable cell density. Detailed Implementation

[0030] The present application will be further described below with reference to specific embodiments.

[0031] 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 this application pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0032] Unless otherwise specified, all materials and reagents used in this article are commercially available.

[0033] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0034] Those skilled in the art will know that "approximately" or "around" refers to numerical values ​​that can achieve the same effect within a certain range.

[0035] Unless otherwise specified in this application, the cells are porcine muscle stem cell lines adapted to carrier-free and serum-free suspension culture by the applicant, named piglet muscle stem cell line YP-S4-S-SC, deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCCNO:C2022372 and deposit date of December 7, 2022. For details, please refer to Chinese Invention Patent Publication No. CN116555171A.

[0036] In this application, unless otherwise specified, cell density refers to the number of cells contained in a unit volume (e.g., per milliliter) of cell suspension, reflecting the density of cells in the culture system.

[0037] In this application, unless otherwise specified, cell viability refers to the proportion of live cells in the total number of cells, usually expressed as a percentage, and is an important indicator for measuring cell health and the suitability of culture conditions.

[0038] Example 1: Test on the effect of cleaning solutions with different additives on cleaning suspended cells Table 1. Types and concentrations of substances added to the cleaning solution.

[0039] The vitamin supplement described in this embodiment comprises 0.005 g / L biotin, 0.005 g / L choline chloride, 0.005 g / L folic acid, 0.01 g / L inositol, 0.005 g / L nicotinamide, 0.005 g / L pantothenic acid•1 / 2Ca, 0.005 g / L pyridoxal hydrochloride, 0.0005 g / L riboflavin, 0.005 g / L thiamine hydrochloride, and 0.425 g / L sodium chloride, which are mixed to form the vitamin supplement.

[0040] The amino acid supplement described in this embodiment comprises 0.1264 g / L arginine hydrochloride, 0.024 g / L cysteine, 0.042 g / L histidine hydrochloride monohydrate, 0.0524 g / L isoleucine, 0.0524 g / L leucine, 0.0725 g / L lysine hydrochloride, 0.0151 g / L methionine, 0.033 g / L phenylalanine, 0.0476 g / L threonine, 0.0102 g / L tryptophan, 0.036 g / L tyrosine, and 0.0468 g / L valine, which are mixed to form the amino acid supplement.

[0041] The components used for washing suspended cells in each experimental group are set as follows: Group 1: Negative control—physiological saline; Group 2: Ultrapure water + 4 common substances in the cleaning solutions of components 1 to 4 shown in Table 1; Group 3: Based on Group 2, add component 5 (polyvinyl alcohol, PVA) as shown in Table 1; Group 4: Based on Group 2, add component 6 (polyethylene glycol, PEG) as shown in Table 1. Group 5: Based on Group 2, add component 7 (methylcellulose, MC) as shown in Table 1; Group 6: Based on Group 2, add component 8 (polyvinylpyrrolidone, PVP) as shown in Table 1. Group 7: Based on Group 2, add component 9 (sodium carboxymethyl cellulose, CMC) as shown in Table 1. Group 8: Based on Group 2, add component 10 (xanthan gum) as shown in Table 1; Group 9: Based on Group 2, add Groups 5 and 10 as shown in Table 1.

[0042] All cleaning solutions to be tested need to have their pH measured and calibrated to approximately 6.9-7.3 and their osmotic pressure adjusted to approximately 290-350 mOsm / kg after preparation.

[0043] (1) Cell harvesting: The cell density in the shake flask is cultured to 8×10⁶ cells / year. 6 When the number of cells / mL is above a certain level, aspirate cells from the shake flask for a washing solution test.

[0044] (2) Cell washing: Take 10 groups of 40 mL cell fluid from the shake flask and put them into 50 mL centrifuge tubes. Centrifuge at 330 g / 5 min and discard the supernatant. Add 4 times the volume of washing solution as the weight of the cell pellet and shake well. Then place the tubes on a shaker to keep the cells in a long-term washing state.

[0045] (3) Cell counting: Samples were taken and counted at two time points, 0.5 h and 3 h. The cell washing effect was judged by observing the changes in cell viability and viable cell density in different washing solution groups.

[0046] The results showed that group 9 had the highest cell density and cell viability after 3 hours, which were significantly higher than other groups. Moreover, in the comparison between 0.5 hours and 3 hours, which represent the washing process, group 9 did not show significant changes in cell density and cell viability during the washing process, while other groups showed significant decreases in cell density and cell viability during the washing process. This indicates that only the washing solution formulation of group 9 can maintain stable cell density and viability during the washing process.

[0047] Example 2: Cell viability, viable cell density, and morphology detection after washing cells with washing solution. (1) Preparation of cleaning solution 1: The recommended composition and concentration of the substances added to cleaning solution 1 in this embodiment are shown in Table 2 below. After preparation, the pH needs to be tested and calibrated to about 6.9~7.3, and the osmotic pressure needs to be adjusted to about 290~350 mOsm / kg.

[0048] Table 2. Composition and Recommended Concentration of Cleaning Solution 1

[0049] (2) Cell viability and viable cell density detection: The suspended cells were washed with washing solution 1. During the cell washing process, the cells were sampled at 4 time points (0.5, 1, 2 and 3 h). 20 μL of cell suspension was stained with 20 μL of trypan blue dye and the cells were counted using a cell counting chamber.

[0050] (3) Cell morphology detection after washing: The suspended cells were washed with washing solution 1 for a long time. Microscopic images were taken before and after washing (3 h after washing) to observe cell morphology.

[0051] The results showed that using washing solution 1 for continuous washing of suspended cells for an extended period of time could maintain cell viability above 90%, and no significant decrease in viable cell density was observed. Figure 3 Compared to before washing, the cell morphology remained stable, and the cells were suspended in the washing solution in a single-cell dispersed state. Figure 4 ).

[0052] Comparative Example 1 This comparative example uses physiological saline to wash the suspended cells.

[0053] Referring to Example 2, the difference is that physiological saline was used instead of washing solution 1 for prolonged washing of the suspended cells, while all other conditions remained unchanged. After prolonged washing of the suspended cells with physiological saline, both cell viability and viable cell density decreased significantly. Figure 5 Morphological observation further revealed that this treatment caused cell rupture, accompanied by the production of a large amount of cell debris. Figure 6 In stark contrast to the cleaning solution 1 in Example 2, this further demonstrates that cleaning solution 1 can be used to clean suspended cells.

[0054] Example 3 Detection of residual substances after cell washing (1) Cell collection: The suspended cells were continuously washed with washing solution 1 in Example 2 for a long time, and the cell precipitate samples were collected by centrifugation at 4 time points for testing.

[0055] (2) Pretreatment: Take the above-collected sample and extract it with a mixed solution of acetonitrile:methanol:water containing 0.1% formic acid and 1mM Na2EDTA in a volume ratio of 1:1:1. After extraction, sonication, centrifugation and filtration, the sample to be obtained is obtained.

[0056] (3) Residue Detection: The test samples were detected by LC-MS / MS under the following chromatographic conditions: Column: LC-MS column with HSS T3 stationary phase or C18 stationary phase as packing material; Mobile phase A: 0.05% formic acid + 10mM ammonium formate water; Mobile phase B: methanol; Flow rate: 0.3 mL / min; Column temperature: 35℃; Injection volume: 10 μL; Mass spectrometry conditions: Ion source type: electrospray ionization source; Scanning mode: selective ion monitoring; Collision activation parameter (CAD): neutral; Curtain gas (CUR): 35 psi; Nebulizer gas (GS1): 50 psi; Auxiliary gas (GS2): 50 psi. psi; Ion source voltage (IS): 5500V / -4500V; Ion source temperature (TEM): 500℃. Compound scanning parameters are: Forsocrine: parent ion 409.3, daughter ion 349.4; declustering voltage -63.6V, collision energy -16.3V; Acetylcysteine: parent ion 164.0, daughter ion 122.0; declustering voltage 20.0V, collision energy 20.0V; Ethanolamine: parent ion 61.9, daughter ion 45.0; declustering voltage 25.0V, collision energy 19.0V.

[0057] The results showed that using washing solution 1 for prolonged continuous washing of suspended cells significantly reduced the levels of residual hazardous substances (forsocolin, acetylcysteine, ethanolamine) in the supernatant and biomass within 1 hour, and continued to decrease over time, eventually reaching an acceptable range. Figure 7 ).

[0058] In summary, the efficient, safe, and low-cost suspended cell washing solution of the present invention can effectively maintain cell viability, cell density, and normal morphology after multiple washes, while efficiently removing residual culture medium, cell debris, metabolic waste, and process-related impurities. This achieves the goal of harvesting high-purity, high-activity, and low-risk substances from suspended cells, and is particularly suitable for the production process of high-requirement cell products such as cultured meat.

[0059] Example 4: Investigation of the Concentration Range of Cleaning Solution To investigate the applicable concentration range of the washing solution for suspended cells of the present invention, this embodiment uses the concentrations of each component in Table 2 above as a benchmark, and simultaneously adjusts the concentrations of the components used to investigate the specific group settings as shown in Table 3.

[0060] Table 3 explores the concentration range of the cleaning solution.

[0061] All cleaning solutions to be tested need to have their pH measured and calibrated to approximately 6.9-7.3 and their osmotic pressure adjusted to approximately 290-350 mOsm / kg after preparation.

[0062] (1) Cell harvesting: The cell density in the shake flask is cultured to 8×10⁶ cells / year. 6 When the number of cells / mL is above a certain level, aspirate cells from the shake flask for a washing solution test.

[0063] (2) Cell washing: Take 40 mL of cell solution from 5 shake flasks and transfer them to 50 mL centrifuge tubes. Centrifuge at 330 g / 5 min and discard the supernatant. Add 4 times the volume of washing solution as the weight of the cell pellet and shake well. Then place the tubes on a shaker to allow the cells to be washed for a long time.

[0064] (3) Cell counting: Samples were taken and counted at two time points, 0.5 h and 3 h. The cell washing effect was judged by observing the changes in cell viability and viable cell density in different washing solution groups.

[0065] The results showed that compared with the existing washing solution group 1, groups 4 and 7 showed significantly reduced cell viability and viable cell density after 0.5 h and 3 h of washing, indicating that the washing solution of this application cannot achieve the desired effect in any proportion. No significant differences were observed in cell viability and viable cell density at the above two time points in the other experimental groups. Figure 8 , Figure 9 Therefore, it can be concluded that the concentration range of each substance added to the washing solution for suspended cells described in this invention is between 2 / 5 and 3 times the concentrations shown in Table 2.

[0066] Example 5: Application of cleaning solution in scale-up production (1) Cell propagation: The YP-S4-S-SC cell line was scaled up step by step from shake flasks to reactors, and finally transferred to a 200 L bioreactor for scale-up culture.

[0067] (2) Cell counting: When the cell density reaches 8×10⁶ cells / year, the cell count is determined by the cell count. 6 When the number of cells / mL is above a certain level, harvest the cells and perform continuous washing and harvesting using the ratios described in Table 2.

[0068] (3) Cell washing and harvesting: Add 4 times the volume of the washing solution to each round of washing and mix gently. Use a butterfly centrifuge with the following centrifugation conditions: centrifugal force 1000g / min, centrifugation temperature 2~8℃. Discard the supernatant washing solution, retain the cell precipitate, and repeat the addition of washing solution until 4 consecutive washings are completed. During the cell washing and harvesting process, cells should be sampled and counted.

[0069] The results showed that in large-scale production, the cleaning solution could maintain cell viability and density during the cleaning and harvesting process. Figure 10 ).

[0070] In summary, the present invention provides a safe, efficient, and low-cost washing solution for suspended cells that maintains cell viability and density, preserves cell morphology, and reduces cell debris during the washing process. This washing solution is adaptable to continuous harvesting processes in large-scale bioreactors, meeting the requirements for cell washing in large-scale production. It enables stable, efficient, and repeatable cell collection and washing operations, satisfying the needs of all scenarios from laboratory research and development to industrial production.

[0071] The examples disclosed above are for illustrative purposes only and should not be construed as limiting the invention. Many different cleaning fluid components are listed herein, and numerous further combinations are possible without departing from the scope and spirit of the invention. Therefore, the invention is not limited to the disclosed embodiments. In fact, all modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A safe, efficient, and low-cost washing solution for suspended cells, characterized in that, The washing solution used for suspended cells includes multiple components such as glucose, amino acid supplements, vitamin supplements, shear protectants, and sodium bicarbonate. The amino acid supplement includes arginine hydrochloride, cystine, histidine hydrochloride monohydrate, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine. The vitamin supplement includes D-biotin, choline chloride, folic acid, inositol, nicotinamide, D-pantothenic acid•1 / 2Ca, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, and sodium chloride. The shear protectant is polyvinyl alcohol and xanthan gum.

2. The washing solution for suspended cells according to claim 1, characterized in that, The total concentration of various substances added to the washing solution used for suspended cells ranges from 9.65156 to 72.3867 mg / mL.

3. The washing solution for suspended cells according to claim 1, characterized in that, The total concentration of various substances added to the washing solution used for suspended cells ranges from 12.06445 to 60.32225 mg / mL.

4. The washing solution for suspended cells according to claim 1, characterized in that, The total concentration of various substances added to the washing solution used for suspended cells ranges from 24.1289 to 60.32225 mg / mL.

5. The washing solution for suspended cells according to claim 1, characterized in that, In the washing solution used for suspending cells, the concentration of any added substance ranges from 0.04 to 60 mg / mL.

6. The washing solution for suspended cells according to claim 1, characterized in that, In the washing solution used for suspended cells, each substance is added at 0.4 to 3 times the following dosage references: glucose 20 mg / mL, amino acid supplement 0.5584 mg / mL, vitamin supplement 0.4705 mg / mL, polyvinyl alcohol 1 mg / mL, sodium bicarbonate 2 mg / mL, and xanthan gum 0.1 mg / mL.

7. The application of the washing solution for suspended cells according to claim 1 in washing suspended cells, characterized in that, The suspended cells are cleaned using the cleaning solution described in claim 1.

8. The application according to claim 7, characterized in that, The cleaning solution described in claim 1 is used to clean the harvested suspended cells in scale-up production.

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