JP2012239444A - Cell culture carrier, method for producing cell culture carrier, and cell culture method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a culture technique by which the interaction between different kinds of cells can be reproduced in a micro environment, and after the interaction, each cell can be collected individually and analyzed sufficiently.SOLUTION: The cell culture carrier 10 includes a hydrogel-dried product obtained by laminating several layers of thin films 12 made of a hydrogel, and drying the laminate. The method for producing the cell culture carrier 10 includes a step of forming the laminate by laminating several layers of the thin films 12 made of the hydrogel, and a step of drying the laminate. The cell culture method includes a step of rehydrating the cell culture carrier 10, a step of culturing first and second cells on both surfaces of the rehydrated cell culture carrier 10, and a step of separating the cell culture carrier 10 into the thin films including the first and the second cells respectively, after the culturing step.

Description

  The present invention relates to a cell culture carrier, a method for producing the same, and a cell culture method using such a cell culture carrier.

  Life activities such as cell proliferation, differentiation, and functional expression are affected by humoral factors in the microenvironment surrounding the cells, cell-cell interactions, and cell-extracellular matrix interactions. Various culture techniques have been established as a method for reproducing the activity of these cells in vitro.

  For example, as a technique for analyzing an interaction between different cells via a humoral factor, a culture method using a container made by Corning, which is called a transwell, is known. In this culturing method, each cell can be collected after co-culture of different cells. However, there is a problem that mimic is difficult when the distance between both cells is far compared with that in the living body and the humoral factor is via the extracellular matrix.

  In addition, as a technique for analyzing direct interaction between different types of cells and interaction between cells and extracellular matrix, a co-culture method for culturing two or more types of cells on the same plane of a plastic culture vessel or gel. A co-culture method in which one of two types of cells is planarly cultured on a gel and the other cell is embedded in a gel, and a co-culture method in which two or more types of cells are embedded in a gel is known. In these culture methods, the cells after interaction cannot be collected separately.

  On the other hand, a solution containing an extracellular matrix is gelled together with a support, dried and vitrified, and then rehydrated and integrated with the support on both sides of an extracellular matrix-containing hydrogel thin film called “vitrigel” In addition, a technique for co-culturing different cells has been devised (see, for example, Patent Document 1). In this culturing method, the interaction between the heterogeneous cells via the extracellular matrix or its substitute, or the interaction via the humoral factor can be reproduced in a finer environment.

  However, in this co-culture method using “Vitrigel”, there are heterogeneous cells on both the front and back sides of one thin film. Therefore, when cells are collected using trypsin after co-culture, each cell is collected individually. The analysis of the interaction between different types of cells is limited.

Japanese Patent No. 3081130

  The present invention has been made to solve the above-mentioned problems of the prior art, and can reproduce the interaction between different types of cells in a fine environment, and collect each cell individually after the interaction. It is an object of the present invention to provide a culture method capable of performing sufficient analysis, a cell culture carrier used in such a culture method, and a method for producing the same.

  The cell culture carrier according to one embodiment of the present invention is characterized by comprising a dried hydrogel obtained by laminating and drying a plurality of thin films made of hydrogel.

  The method for producing a cell culture carrier according to another aspect of the present invention includes a step of laminating a plurality of thin films made of hydrogel to form a laminate, and a step of drying the laminate.

  The cell culture method according to still another aspect of the present invention includes a step of rehydrating the cell culture carrier and a step of culturing the first and second cells on both sides of the rehydrated cell culture carrier. And a step of separating the cell culture carrier into thin films containing the first and second cells, respectively, after the cell culturing step.

  In the present specification, “hydrogel” refers to a gel-like substance in which a polymer has a network structure formed by chemical bonds and a large amount of water is retained in the network.

  According to the present invention, interaction between different types of cells can be reproduced in a fine environment, and after the interaction, each cell can be individually collected and sufficient cell analysis can be performed. become.

It is a schematic sectional drawing which shows the cell culture support | carrier of one Embodiment of this invention, and its manufacturing method. It is a top view of the cell culture support | carrier shown in FIG. It is sectional drawing explaining an example of the cell culture method using the cell culture carrier shown in FIG.

  Embodiments of the present invention will be described below. Although the description will be made based on the drawings, the drawings are provided for illustration only, and the present invention is not limited to the drawings.

  FIG. 1 is a schematic cross-sectional view showing a cell culture carrier and a manufacturing method thereof according to one embodiment of the present invention, and FIG. 2 is a plan view showing the cell culture carrier of this embodiment.

  As shown in FIG. 1, the cell culture carrier 10 of the present embodiment is obtained by laminating and drying a thin film 12 made of two hydrogels.

  The polymer material constituting the thin film 12 made of hydrogel may be derived from a natural product or synthesized. Examples of polymer materials derived from natural products include collagen, laminin, fibronectin, vitronectin, nidogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, proteoglycan, Matrigel (trade name; from mouse EHS tumor extract) Examples include extracellular matrix components such as a reconstituted basement membrane component), gelatin, agar, and agarose. Examples of the synthetic polymer material include polyacrylamide, polyvinyl alcohol, methyl cellulose, polyethylene oxide, poly (2-hydroxyethyl acrylate) / polycaprolactone, and the like.

  In a polymer material derived from a natural product, a hydrogel can be produced by selecting a salt component suitable for each gelation, its concentration, pH, and the like. For example, in the case of collagen, PBS (Phosphate Buffered saline), HBSS (Hank's Balanced Salt Solution), a basic culture solution, a serum-free culture solution, a serum-containing culture solution, or the like is used as a solvent that provides a suitable salt concentration. .

  In the present invention, as the thin film 12 made of hydrogel, a hydrogel that has been dried and vitrified and then rehydrated can be used. Here, “vitrification” means vitrification technology (Takushi E.,) which is obtained by heat-denaturing chicken egg protein (white) (hydrogel) into a hard and transparent substance by air drying. (Edible eyeballs from fish. Nature 345, 298, 1990). The thin film 12 may be provided with a support 14 made of, for example, nylon resin for maintaining the shape. Providing the support 14 facilitates handling of the thin film 12, and thus the cell culture carrier 10, and facilitates separation of the thin film 12 after cell culture.

  In this embodiment, as a thin film 12 made of hydrogel, a collagen solution as an extracellular matrix component is gelled together with an annular support made of nylon resin, dried and vitrified, and then rehydrated and supported. A “thin film made of collagen gel” integrated with the body (hereinafter also referred to as “collagen vitrigel”) is used.

Hereinafter, the collagen vitrigel used in the present invention will be described.
Any type of collagen, such as type I, type II, type III, and type V, can be used as long as it can be gelled, but type I collagen is preferably used.

  The solvent used for dissolving collagen is not particularly limited as long as it does not dissolve collagen and alter the collagen. Specifically, for example, water, hydrochloric acid solution, methyl alcohol, ethyl alcohol, phosphate buffer, and a mixture thereof can be used.

  Gelatinization of collagen can be performed by preparing collagen in a solution having an appropriate salt concentration and pH, preferably pH 6 to 10, and maintaining the solution at an appropriate temperature, preferably 37 ° C.

  As a method for drying the collagen gel, methods such as air drying, drying in a sealed container (circulating air in the container and always supplying dry air), drying in an environment where silica gel is placed, and the like can be used. Examples of the air drying method include a method of drying in an incubator kept sterile at 10 ° C. and 40% humidity for two days, or drying at room temperature all day and night in a sterile clean bench. After air drying, store aseptically at room temperature or at 4 ° C while maintaining aseptic conditions. It has been confirmed that when the storage time is one month or more, the strength and transparency are significantly increased.

  In addition, the time for drying the collagen gel varies depending on the moisture content and the like, but as a result of examining changes in compressive fracture strength, transparency, and mass, the mass after drying is 1/50 to 1/100 of the mass of the collagen gel. By setting the drying time so as to decrease, a thin film excellent in strength and transparency (hereinafter also referred to as “collagen vitrigel dried body”) can be produced.

  The collagen vitrigel is obtained by rehydrating the “dried collagen vitrigel”. The collagen vitrigel preferably has a thickness in the range of 1 μm to 1 mm. If the thickness is thinner than 1 μm, the strength is insufficient. On the other hand, a collagen vitrigel having a thickness exceeding 1 mm can be obtained by increasing the amount of collagen gel before drying, but it takes a long time to dry.

Further, collagen Vito Rigel, it is preferable to the collagen content per unit area in the range of ^{100μg / cm 2 ~1mg / cm 2} , and more preferably in the range of ^{150μg / cm 2 ~500μg / cm 2} . When the collagen content is less than 100 μg / cm ² , the collagen concentration is too thin, the gelation is weak, and the strength is insufficient.

  The collagen vitrigel can contain a physiologically active substance in addition to collagen as a gel component. Examples of physiologically active substances include cell growth factors, differentiation-inducing factors, cell adhesion factors, antibodies, enzymes, cytokines, hormones, lectins, and non-gelling extracellular matrix components such as fibronectin, vitronectin, entactin, and osteopoietin. Can be mentioned. A plurality of these can be contained.

  Collagen vitrigel containing a bioactive substance is mixed with a gel-constituting polymer solution before gelation, that is, a collagen solution, and then the bioactive substance to be contained is mixed. It can be manufactured through a manufacturing process. A collagen vitrigel containing a physiologically active substance can supply factors necessary for cell growth, differentiation, adhesion, and the like from the collagen vitrigel side, so that a better culture environment can be realized. It is also useful in conducting tests for examining the effect of the contained physiologically active substance on cells.

  The cell culture carrier 10 is produced by laminating and drying a thin film 12 made of a hydrogel such as the collagen vitrigel.

  As a drying method after laminating the thin film 12, a method similar to the drying method in producing the collagen vitrigel can be used. That is, methods such as air drying, drying in an airtight container (circulating air in the container and always supplying dry air), and drying in an environment where silica gel is placed can be used. Examples of the air drying method include a method of drying in an incubator kept sterile at 10 ° C. and 40% humidity for two days, or drying at room temperature all day and night in a sterile clean bench. After air drying, store aseptically at room temperature or at 4 ° C while maintaining aseptic conditions. Similarly to the case of preparing a dried collagen vitrigel, when the storage period is set to 1 month or more, the strength and transparency can be increased.

  The drying time is preferably set to a time when the moisture content after drying is equivalent to the atmosphere during drying. Furthermore, it is preferable to set the drying time and the drying atmosphere so that the water content after drying is 30% by mass or less. By setting in this way, it has sufficient strength and transparency that does not peel at the interface of the thin film 12, and after rehydration in use, it can be easily peeled off at the interface of the thin film 12 as necessary. A cell culture carrier 10 that can be produced can be produced.

  In the cell culture carrier 10 of this embodiment, as shown in FIG. 2, notches 16a and 16b are slightly shifted in the circumferential direction on the inner and outer circumferences of the annular support 14 provided on each thin film 12. The front and back of each thin film 12 can be identified by the relative positions of the inner notch 16a and the outer notch 16b. By making the front and back of the thin film 12 distinguishable in this way, when the thin film 12 is separated, the side on which the cells are cultured can be easily known. The front and back sides can be identified by, for example, printing an identification mark on at least one of the front surface and the back surface of the support 14 without depending on the notches 16a and 16b. However, from the viewpoint of influence on cell culture, ease of formation, etc., discrimination by notch formation as in this embodiment is preferable. The shape of the notch is not particularly limited as long as the function as a support is not impaired.

  The cell culture carrier 10 of this embodiment can be used as a cell culture carrier for culturing animal cells. Examples of animal cells to be cultured include primary cultured cells, established cells, fertilized eggs, and cells obtained by introducing a foreign gene into these cells. These may be undifferentiated stem cells, cells in the process of differentiation, terminally differentiated cells, or dedifferentiated cells. Means for initiating the culture of these cells include seeding of cell suspensions, minced tissue pieces, fertilized eggs, or three-dimensionally reconstructed multicellular aggregates. That is, any of the adherent cells that can be cultured by an existing method can be cultured on the present cell culture carrier 10.

  In addition, the above-described animal cells can be cultured on one side of the cell culture carrier 10, or one or more types of cells can be cultured on both sides. In addition, the cell culture carrier 10 of this embodiment is particularly useful. For example, by culturing epithelial cells on one side and mesenchymal cells on the other side, cell function assays such as percutaneous absorption model and intestinal absorption model with epithelial-mesenchymal interaction are possible. In addition, by culturing vascular endothelial cells on one surface and cancer cells on the other surface, cell function assays such as an angiogenesis model and a cancer invasion model are possible.

    Furthermore, the reconstructed body having an epithelial-mesenchymal interaction cultured using the cell culture carrier 10 of the present embodiment can be applied to an alternative model for animal experiments, development of a cultured organ, and transplantation of a cultured organ. is there. The cell culture carrier 10 alone can be used to prevent organ adhesion. However, after culturing cells on one or both sides of the cell culture carrier 10 and seeding corneal epithelial cells taking advantage of the transparency, collagen vitrigel is used. With the method of transplanting to patients with corneal defects or aging society, peritoneal mesothelial tissue has been lost due to several laparotomy, causing problems such as intestinal organ adhesion and complications such as intestinal obstruction. On the other hand, it is possible to culture not only the peritoneum but also the pleura and pericardial mesothelium on the present cell culture carrier 10 and transplant an excellent culture carrier that secretes lubricating components (such as hyaluronic acid).

  As described above, the cell culture carrier 10 of this embodiment is particularly useful for double-sided culture in which different types of cells are cultured on each side. Hereinafter, an example of a method for culturing different types of cells on both surfaces of the cell culture carrier 10 will be described.

  (1) First, PBS, HBSS, a basic culture solution, a serum-free culture solution, a serum-containing culture solution, etc. are added to the cell culture carrier 10 that has been dried in a culture vessel such as a petri dish and stuck to the bottom of the vessel. The cell culture carrier 10 is rehydrated by allowing it to stand for several minutes.

  (2) When the cell culture carrier 10 is sufficiently rehydrated, PBS and the like are removed, and a culture solution in which cells to be attached (first cells) are suspended is added and cultured. The medium is appropriately selected depending on the type of cells to be used, and is not particularly limited. For example, Dulbecco's MEM medium, BME medium, MCDB-104 medium and the like are used. If necessary, additives such as serum proteins such as serum and albumin, various vitamins, amino acids and salts may be added to the medium. The culture temperature is appropriately selected depending on the type of cells to be cultured and is not particularly limited, but is usually 4 to 40 ° C, preferably 25 to 37 ° C, more preferably 25 to 37 ° C. The culture is continued while appropriately changing the medium.

  (3) After cell culture, the medium is removed from the culture vessel, and the cell culture carrier 10 is taken out together with the cultured cells using tweezers or the like. After removing the medium, it is preferable to wash the surface of the cells with PBS or a medium before taking out the cell culture carrier 10, and after removing the cell culture carrier 10 together with the cells, similarly, with PBS or the medium. It is preferable to wash the cell culture carrier 10 to which the cells adhere.

  (4) Next, the cell culture carrier 10 is allowed to stand on the culture disk of a petri dish for double-sided culture equipped with a culture disk with a culture medium in the center, with the cell culture surface facing down, and then the cells A culture solution in which cells (second cells) to be attached to the entire upper surface of the culture carrier 10 are suspended is added and incubated at 37 ° C., for example.

  (5) When the second cell adheres to the upper surface of the cell culture carrier 10, it is placed in a separately prepared culture container such as a petri dish and the culture is continued in the same manner as in (2). Thereby, the cell culture carrier 10 in which different types of cells are cultured on both sides is obtained.

  (6) Thereafter, only the thin film 12 on which the second cells are cultured is removed from the upper surface of the cell culture carrier 10 so as to be peeled off. This operation can be easily performed using tweezers or the like. As a result, the cell culture carrier 10 in which the first and second cells are cultured on both sides can be separated into the thin film 12 in which the first cells are cultured and the thin film 12 in which the second cells are cultured. The cell observation suitable for each can be performed. In addition, the first and second cells can be recovered from the separated thin film 12 using a stripping solution such as trypsin, dispase, actase, EDTA, etc., and used for various experiments and research. FIG. 3 is a diagram schematically showing a state in which the cell culture carrier 10 in which the first cell 21 and the second cell 22 are cultured on both surfaces is separated into two thin films 120.

  Thus, in the double-sided culture method using the cell culture carrier 10 of the present embodiment, different types of cells can be individually observed and recovered without being influenced by the other cell. Therefore, it becomes possible to perform a deeper and more accurate analysis of the interaction between different types of cells.

  In the above-described embodiment, a cell culture carrier having a two-layer structure in which two thin films are stacked has been described. However, a three-layer or more multilayer structure in which three or more thin films are stacked may be used. Needless to say, you can. For example, a cell culture carrier having a three-layer structure in which three thin films are laminated can be obtained by first cultivating the first and second cells on both sides and then separating only the thin film to which the first cells are attached. The third cells can be seeded and cultured on the exposed surface, and the first cells and the second cells affected by the third cells can be observed or collected.

  The thin film to be laminated may be formed of the same kind of polymer material, or may be formed of a different polymer material. For example, a thin film made of Matrigel is laminated on a thin film made of collagen gel. Also good.

  EXAMPLES Next, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples at all.

[Example 1: Cell culture carrier]
(Preparation of dried collagen vitrigel)
An annular (circular hollow) support having an outer diameter of 33 mm and an inner diameter of 24 mm was prepared by cutting out a nylon membrane (trade name Amersham # RPN1782B, manufactured by Amersham), and after sterilization, a hydrophobic polystyrene culture petri dish (φ35 mm; Asahi Glass ( Co., Ltd. product name IWAKI # 1000-035). In a 50 ml sterile conical tube (trade name IWAKI # 2342-050 manufactured by Asahi Glass Co., Ltd.) cooled on ice, 2.5 ml of cell culture solution {10% non-immobilized fetal bovine serum (FBS), 20 mmol / l HEPES (Life Dulbecco's modified Eagle medium (trade name Mediatech # 10-017-CV, manufactured by Mediatech Co., Ltd.) and 2.5 ml of 0.5% type I collagen aqueous solution (trade name), manufactured by Technologies, Inc., trade name GIBCO # 15630-080)} Kenken brand name Koken # IAC50) was added and mixed uniformly. 1.0 ml of a collagen mixture solution having a final concentration of 0.25% was placed in a hydrophobic polystyrene culture dish containing the above support, and then a 37 ° C. moisturizing incubator in the presence of 5% CO ₂ /95% air. And gelled for 2 hours. This final concentration 0.25% collagen gel was vitrified by aseptically drying for 2 days with the lid removed in a clean bench. 3 ml of PBS was added to the vitrified collagen gel and rehydrated. After rinsing twice with 3 ml of PBS, it was aseptically dried for 2 days with the lid removed in a clean bench to obtain dried collagen vitrigel A.

(Production of collagen vitrigel multilayer)
1 ml of each sterilized water was added to the two dried collagen vitrigel A and rehydrated to obtain a collagen vitrigel. One collagen vitrigel was peeled off from the petri dish using tweezers and overlaid on the other collagen vitrigel. Remove the sterilized water so that no gas enters between the two collagen vitrigels, and then aseptically dry for 2 days with the lid removed in a clean bench, to obtain the dried collagen vitrigel multilayer body. Obtained.

(Collagen Vitrigel Multilayer Separation Confirmation Test)
The obtained dried collagen vitrigel multilayer body was placed in 2 ml of a culture solution and allowed to stand for 5 minutes for rehydration. By sandwiching the two rehydrated collagen vitrigel multilayer substrates with tweezers while overlapping them, the multilayer body could be peeled off from the culture vessel. Furthermore, when each support body of this collagen vitrigel multilayer body is pinched with tweezers and peeled off, one collagen vitrigel multilayer body can be easily separated into two collagen vitrigels without damaging them. It was.

(Protein permeability test of collagen vitrigel multilayer)
Also, 30% FBS / PBS is placed in one column of a membrane permeation experiment apparatus PERMCELL (product name) manufactured by Beadrex, and PBS is placed in the other column. The collagen vitrigel multilayer body is placed in the center of both columns. Samples were collected over time from the column on the PBS side. The collected sample was electrophoresed and then silver-stained, and protein permeability through the collagen vitrigel multilayer was examined. From the first day, permeation of a protein having a large molecular weight exceeding 110 kDa was confirmed.

  For comparison, in the method for preparing a dried collagen vitrigel described above, the amount of the 0.25% collagen mixed solution to be placed in the hydrophobic polystyrene culture petri dish was changed from 1.0 ml to 2.0 ml. The same protein permeability test was carried out on a single-layered collagen vitrigel obtained by rehydrating the dried collagen vitrigel B prepared in the same manner, and protein permeation was confirmed on the first day. Permeation of proteins exceeding 110 kDa was seen with the eyes.

  From these results, it was found that the collagen vitrigel multilayer body has the same protein permeability as the collagen vitrigel having the same thickness.

[Example 2: Cell culture using cell culture carrier]
3 ml of PBS was added to the dried collagen vitrigel multilayer body produced in Example 1 and left for 5 minutes to rehydrate. After removing the excess PBS, one surface of the rehydrated collagen vitrigel multilayer was seeded with 2 ml of CACO-2 cell suspension (1 × 10 ⁵ cells / ml) stained with PKH26, The cells were cultured in a 37 ° C. moisturizing incubator in the presence of 5% CO ₂ /95% air. After culturing for 4 days, the inner wall of the petri dish was traced with tweezers, and the collagen vitrigel multilayer body in which CACO-2 was cultured on one side was peeled off and collected. The collected collagen vitrigel multilayer body is a Dulbecco containing 3 ml of a culture solution ({10% non-immobilized fetal bovine serum (FBS), 20 mmol / l HEPES (trade name GIBCO # 15630-080) manufactured by Life Technologies)} in the central portion. A cell culture surface is placed on a disk for double-sided culture (trade name: Collagen Gel Thin Film Vitrigel # VIT-C001, manufactured by Asahi Glass Co., Ltd.) containing a modified Eagle medium (trade name Mediatech # 10-017-CV, manufactured by Mediatech). Placed on the bottom and seeded with human dermis-derived fibroblasts (4 × 10 ⁵ cells / 350 μl) stained with PKH2 on the top surface, in a 37 ° C. humidified incubator in the presence of 5% CO ₂ /95% air For 2 hours. Thereafter, Dulbecco's modified Eagle medium (trade name Mediatech, manufactured by Mediatech Co., Ltd.) containing 2 ml of culture medium ({10% non-immobilized fetal bovine serum (FBS), 20 mmol / l HEPES (trade name: GIBCO # 15630-080, manufactured by Life Technologies)) # 10-017-CV)) was added to a non-surface-treated petri dish (φ35 mm: IWAKI # 3000-035X), and the collagen vitrigel multilayer body in which cells were seeded on both sides was transferred to the above and Similarly, it was cultured for 3 days. Then, the collagen vitrigel multilayer body in which cells were seeded on both sides was separated into two thin films using tweezers, and each was observed with a phase contrast microscope and a fluorescence microscope. Only cells and only human dermis-derived fibroblasts on the other thin film were observed.

  The cell culture carrier obtained by the present invention can reproduce the interaction between different types of cells in a fine environment, and can collect each cell individually after the interaction. It is useful for analysis of action.

  DESCRIPTION OF SYMBOLS 10 ... Cell culture support | carrier, 12 ... Thin film which consists of hydrogels, 14 ... Support body.

Claims (9)

  A cell culture carrier comprising a dried hydrogel obtained by laminating and drying a plurality of thin films made of hydrogel.   2. The cell culture carrier according to claim 1, wherein the thin film made of hydrogel is obtained by drying and vitrifying the hydrogel and then rehydrating it.   The cell culture carrier according to claim 1 or 2, wherein the thin film made of each hydrogel is supported by a support.   The cell culture carrier according to any one of claims 1 to 3, wherein the thin film made of each hydrogel contains an extracellular matrix component.   The thin film comprising each hydrogel contains at least one extracellular matrix component selected from collagen, fibronectin, vitronectin, laminin, nidogen, tenascin, thrombospondin, proteoglycan and fibrinogen. 4. The cell culture carrier according to any one of 3.   The cell culture carrier according to any one of claims 1 to 3, wherein the thin film made of hydrogel is a thin film mainly composed of collagen.   The cell culture carrier according to any one of claims 1 to 6, wherein the cell culture carrier can be separated into a thin film composed of a plurality of hydrogels by rehydration. A step of laminating a plurality of thin films made of hydrogel to form a laminate;
And a step of drying the laminate. A method for producing a cell culture carrier. Rehydrating the cell culture carrier according to any one of claims 1 to 7,
Culturing the first and second cells respectively on both sides of the rehydrated cell culture carrier;
Separating the cell culture carrier into thin films containing the first and second cells, respectively, after the cell culturing step.

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