KR102644816B1 - A preparation device of biological product using a micro bioreactor and preparation method therewith - Google Patents

Abstract

In the biological agent production device according to the present invention, cell aggregates are located in a hollow spherical hydrogel structure, and a plurality of cell aggregates are physically spaced apart by the hydrogel structure, so that during the secretion or production of biological agents, the cell aggregates are It can effectively block secondary aggregation that fuses with each other. Due to this, there is an effect that biological agents such as extracellular vesicles, cell secretion factors, exosomes, antibodies, or viral vectors secreted or produced by cell aggregates, including exosomes, can be continuously and stably manufactured. Due to the presence of a single cell aggregate in a hydrogel structure with a hollow spherical structure, various biological agents produced by the cell aggregate penetrate the hydrogel structure with a porous structure and are easily discharged to the outside of the hydrogel structure, thereby enabling the production of biological agents. and can be easily obtained.

Description

{A preparation device of biological product using a micro bioreactor and preparation method therewith}

The microbioreactor according to the present invention includes cell aggregates located within a hydrogel structure having a hollow sphere shape, and by culturing the microbioreactor in a static or dynamic manner, the cell aggregates within the hydrogel structure can react with various biological agents. It is produced or secreted, and the biological agent produced or secreted in this way is discharged from the inside of the hydrogel structure having a porous structure to the outside, and various biological agents can be stably produced.

[Assignment identification number] S3283917

[Ministry Name] Ministry of SMEs and Startups

[Research Management Institute] Small and Medium Business Technology Information Promotion Agency

[Research Project Name] Small and Medium Business Technology Development Support Project

[Research project title] Development of stem cell spheroid-derived exosome-based wound treatment agent

[Contribution rate] 70/100

[Host organization] Spebio Co., Ltd.

[Research period] 2022.07.01. ~ 2024.06.30.

[Assignment identification number] S3197954

[Ministry Name] Ministry of SMEs and Startups

[Research Management Institute] Small and Medium Business Technology Information Promotion Agency

[Research Project Name] Startup Growth Technology Development Project (TIPS Project)

[Research project name] High-efficiency extracellular vesicle production and development of extracellular vesicle treatment technology

[Contribution rate] 30/100

[Host organization] Spebio Co., Ltd.

[Research period] 2021.10.01. ~ 2023.09.30

Most cellular structures that make up the human and animal bodies are organized in three-dimensional forms. The three-dimensional structure of these cells results in complex interactions between cells that are difficult to mimic in two-dimensional monolayer cell cultures.

In general, living cells behave more naturally when cultured in a three-dimensional environment, but the formation of three-dimensional cell cultures is difficult and expensive in many cases. Conventional in vitro cell culture can only provide a typical two-dimensional cell culture model.

Cellular spheroids are an example of a typical three-dimensional cell culture model, are used in various basic research and clinical pharmacology, and can secrete or produce various biological agents, including exosomes. These cell aggregates are clusters of cells (primary aggregation) and have a diameter of about several hundred micrometers.

Existing methods for producing cell aggregates include dynamic culture methods such as the hanging drop method or rotary agitation. It may take a long period of time, about 4 days, to form cell aggregates using this method, and in practice, The success rate also remains at around 50%. Additionally, it is difficult to exchange the cell culture medium, so there is a problem that cell loss occurs during the exchange process.

Meanwhile, in the case of cell aggregates prepared by this method, static or dynamic culture processes are performed to produce biological products such as exosomes. During this culture process, the characteristics of living cells and physical contact between cell aggregates As a result, a secondary aggregation phenomenon occurs in which a plurality of individual cell aggregates fuse with each other to form a single cell aggregate with a large volume (see Figure 1), and the secretion or production efficiency of biological agents is significantly reduced. It happens.

Here, secondary aggregation is a different concept from primary aggregation, in which individual cells aggregate to form cell aggregates. In a state where cells have already aggregated to form cell aggregates and can produce or secrete various biological agents, individual cells This means that aggregates fuse together for various reasons, such as physical contact, to form a large single cell aggregate of much larger volume (see Figure 1).

The increase in volume due to aggregation during the culture process of these individual cell aggregates causes a shortage of nutrients to the cells inside the increased size cell aggregates, making smooth secretion or production of biological agents difficult. As a result, there is a problem in that cell aggregates exist individually and the amount of secreted or produced biological agent is reduced compared to the amount of biological agent secreted or produced.

In the present invention, in the process of culturing these cell aggregates, in order to fundamentally block physical contact and prevent size increase due to fusion of cell aggregates, microreactors individually located within the hollow spherical hydrogel structure are used to achieve effective biological The production of the formulation was made possible.

Registered Patent No. 2401810 Registered Patent No. 2246224

In the biological agent production device according to the present invention, cell aggregates are located in a hollow spherical hydrogel structure, and a plurality of cell aggregates are physically spaced apart by the hydrogel structure, so that during the secretion or production of biological agents, the cell aggregates are separated. It can prevent cells from fusing together to form a single cell aggregate with a large volume. Through the hollow sphere-shaped hydrogel structure that can prevent the fusion of these cell aggregates, extracellular vesicles, cell secretion factors, exosomes, antibodies, or viral vectors, including exosomes, secreted or produced by cell aggregates. Biological products can be manufactured consistently and reliably on a continuous basis.

In addition, in the micro-bioreactor according to the present invention, a single cell aggregate exists in a hydrogel structure having a hollow spherical structure, so that various biological agents produced by the cell aggregate penetrate the hydrogel structure having a porous structure and enter the hydrogel structure. is discharged to the outside, so the production and acquisition of biological agents can be easily performed. Accordingly, the present invention is intended to provide a microbioreactor suitable for mass production of various biological agents and a method for mass production of biological agents using the same.

The biological agent production device according to the present invention includes a microbioreactor in which cell aggregates are located within a hydrogel structure.

The cell aggregates and hydrogel structures constituting the microbioreactor are spaced apart from each other, so that a space may exist between the cell aggregates and the hydrogel structure, and the hydrogel structures are preferably in the form of hollow spheres. .

Biological agents produced or secreted from single cell aggregates located within the hydrogel structure can be discharged to the outside of the hydrogel structure, and it is more preferable that the cell aggregates located within the hydrogel structure are single cell aggregates.

The biological agent may be an extracellular vesicle, a cell secretion factor, an exosome, an antibody, or a viral vector, and cell aggregates capable of secreting or producing such a biological agent include human-derived cells, animal cells, plant cells, and genetically modified cells. Or, multiple host cells are aggregated.

The hydrogel structure is an in-situ forming or rapid forming hydrogel, and is preferably a polysaccharide hydrogel or protein-based hydrogel crosslinked by ions.

The polysaccharide hydrogel is at least one selected from alginate, chitosan, pectin, and poly(N-isopropyl acrylamide), and the protein-based hydrogel is at least one selected from collagen, gelatin, and silk. It is desirable.

The ions used for crosslinking the polysaccharide hydrogel are preferably divalent or trivalent metal cations such as Fe ³⁺ , Al ³⁺ , Cr ³⁺ , Cu ²⁺ , Ba ²⁺ , Sr ²⁺ , and Ca ²⁺ And, the cross-linking agent used to cross-link the protein-based hydrogel is more preferably genipin, lysyl oxidase, transglutaminase, tyrosinase, or horseradish peroxidase.

The porosity of the hydrogel structure is preferably 90% or more and less than 99%, and in the case of the ion cross-linked hydrogel structure, the concentration of alginate used is preferably 0.1 wt% or more and less than 2 wt%.

Another embodiment of the present invention includes a method for producing a biological agent, wherein a microbioreactor in which cell aggregates are located inside a hollow spherical hydrogel structure is cultured in a culture medium, and the cells located in the hydrogel structure are cultured in a culture medium. Biological agents produced or secreted from aggregates are discharged to the outside through the hydrogel, and the hydrogel structure is an immediate binding reaction hydrogel (in-situ forming) of a polysaccharide hydrogel or protein-based hydrogel cross-linked by ions. or rapid forming hydrogel).

It is preferable that a space is formed between the cell aggregate and the hydrogel structure, and it is more preferable that the cell aggregate located inside the hydrogel structure is a single cell aggregate.

The microbioreactor can produce various biological agents by static or dynamic culture in a culture medium.

The microbioreactor according to the present invention places cell aggregates on each hollow sphere-shaped hydrogel structure, so that a plurality of cell aggregates fuse with each other to form large cell aggregates during the culture process of producing biological agents such as exosomes. Secondary aggregation can be prevented.

Since individual cell aggregates are physically separated from each other by the hydrogel structure, it prevents secondary aggregation of cell aggregates from decreasing the productivity of biological products and the death of cells located inside the secondary aggregates. This has the effect of allowing cell aggregates to produce biological agents stably for a long period of time.

In addition, in the micro bioreactor according to the present invention, since the hydrogel forming the hydrogel structure with a hollow spherical structure has a porous structure, various biological agents produced or secreted by cell aggregates located within the hydrogel structure are porous. By penetrating the hydrogel structure and being discharged to the outside of the hydrogel structure, there is an advantage that the production and acquisition of biological agents can be easily performed and that it is suitable for mass production of various biological agents.

In addition to the obvious effects of the microbioreactor according to the present invention, there are various effects and advantages that can be easily recognized by those skilled in the art from the detailed description of the invention described below.

Figure 1 shows the results of observing secondary aggregation of multiple cell aggregates into one bulky cell aggregate during the culture process over time.
Figure 2 shows that in the microbioreactor 100 according to an embodiment of the present invention, a single cell aggregate 300 is located within the hollow spherical hydrogel structure 200, and the biological cells secreted or produced by the cell aggregate during the culture process are shown in Figure 2. It schematically shows the process by which an agent (400, exosome, antibody, virus, etc.) penetrates the porous hydrogel and moves out of the hydrogel structure.
Figures 3(a) to 3(d) show the change in concentration of alginate, a polysaccharide hydrogel cross-linked by ions (a: 0.1 wt%, b: 0.5 wt%, c: 1.0 wt%, d: 2.0 wt%). This is an observation of the thickness of the porous alginate hydrogel, and the black circle in the middle is a cell aggregate.
Figure 4 shows the results of measuring the change in porosity (%) of the porous alginate hydrogel according to the alginate concentration observed in Figures 3(a) to 3(d).
Figure 5 shows the change in concentration of biological agent measured outside the hydrogel according to the change in porosity of the porous alginate hydrogel observed in Figures 3 and 4, based on the case without the hydrogel structure (100). It shows the converted relative value.

Before explaining in detail the preferred embodiments of the present invention below, the terms or words used in the claims of this specification should not be construed as limited to their usual or dictionary meanings, but should be interpreted as meanings consistent with the technical idea of the present invention. Please note that it should be interpreted as a concept.

Throughout this specification, when a part “includes” a certain component, this means that it may further include other components rather than excluding other components, unless specifically stated to the contrary.

Below, we will look at the present invention in more detail, focusing on embodiments. However, the scope of the present invention is not limited to the preferred embodiments below, and those skilled in the art can implement various modifications of the contents described in this specification within the scope of the present invention.

As schematically shown in FIG. 2, the biological agent production device according to an embodiment of the present invention is a process in which a plurality of living cells such as human-derived cells, animal cells, plant cells, genetically modified cells, or host cells are first aggregated. The formed cell aggregate 300 exists within the hollow sphere-shaped hydrogel structure 200 to form one micro-bioreactor 100.

That is, the microbioreactor (!00) according to the present invention includes a hydrogel structure 200 having a hollow sphere shape and a cell aggregate (spheroid, 300) located inside the hydrogel structure.

Preferably, a single cell aggregate 300 is located within the hydrogel structure 200, so that different cell aggregates 300 are physically spaced apart from each other during a static or dynamic culture process and have a structure in which they cannot contact each other. In Figure 2, it is shown as an example that these microbioreactors 100 secrete or produce biological agents through dynamic culture, but they are not necessarily limited to dynamic culture, and existing known culture methods including static culture methods are used. can be selected

The hydrogel structure 200, which has a hollow sphere shape, is a porous hydrogel formed through an immediate binding reaction ( in-situ forming or rapid forming), and has such a porous structure, forming a cell aggregate (300) located inside the hollow sphere. ) secreted or produced by biological agents (biologics, 400) can move from the inside of the hollow sphere to the outside.

By cultivating this micro-bioreactor 100 statically or dynamically, the cell aggregate 300 can produce various biological agents (biologics, 400), and the produced biological agents 400 can move to the outside of the hydrogel structure 200. You can.

The biological agent 400 may vary depending on the type of cells constituting the cell aggregate 300. For example, the biological agent 400 includes exosomes, antibodies, viruses, and secretome factors. ), etc., and the cell secretion factor refers to a set of proteins expressed by an organism and secreted into the extracellular space, and may include various proteins such as cytokines, growth factors, extracellular matrix proteins, and regulators. You can.

The cell aggregate 300 is located within a hydrogel structure 200 having the shape of a hollow sphere, and it is preferable that a single cell aggregate 300 exists within one hydrogel structure 200. As one cell aggregate 300 is located within the individual hollow sphere-shaped hydrogel structure 200, the individual cell aggregates 300 are prevented from physically contacting each other during the cultivation process of the microbioreactor 100, and secondary The problem of reduced productivity of the biological agent 400 due to aggregation (see FIG. 1) can be effectively solved.

In addition, the cell aggregate 300 is located inside the hydrogel structure 200 and is spaced apart from the inner wall of the hydrogel structure 200 at a predetermined distance, so that a certain space is maintained between the cell aggregate and the hydrogel structure. It can exist. On the other hand, by minimizing the separation distance or space, it is possible to configure the micro-bioreactor 100 in a form such that the hydrogel structure 200 is coated around the cell aggregate 300.

The hydrogel structure 200 of this micro bioreactor 100 can be formed through an immediate bonding ( in-situ forming or rapid forming) reaction. Hydrogels that can be formed through this immediate bonding reaction include, Polysaccharide in which ionic components (e.g. divalent or trivalent metal cations such as Fe ³⁺ , Al ³⁺ , Cr ³⁺ , Cu ²⁺ , Ba ²⁺ , Sr ²⁺ , Ca ^2+, etc.) act as a crosslinking agent (Polysaccharide) hydrogel and protein-based hydrogel in which genipin, lysyl oxidase, transglutaminase, tyrosinase, and horseradish peroxidase act as cross-linking agents.

Representative ionic cross-linked hydrogels include alginate, chitosan, pectin, and Poly (N-isopropyl acrylamide, PNIPAm), and protein-based hydrogels include collagen, gelatin, and silk.

As an example, a method of manufacturing a microbioreactor (100) in which a cell aggregate (300) is located within a hollow sphere-shaped hydrogel structure (200) using an ionic cross-linking method is described as an example. Cell aggregates (300) are primary aggregates of cells. ) is mixed with an ionic solution (e.g., calcium ion) as a cross-linking agent, and then discharged into a hydrogel that can be cross-linked with ions such as alginate through a pipette, dispenser, or 3D printer.

As the discharged ionic solution reacts and crosslinks in situ with the hydrogel capable of ionic crosslinking, an ionic crosslinked porous hydrogel porous membrane is formed on the outer periphery of the discharged ionic solution, and the overall shape has a hollow sphere shape.

It is possible for the ionic solution to contain a large number of cells instead of the cell aggregate (300), and the cells are primarily aggregated inside the hollow sphere-shaped hydrogel formed through this in-situ ionic cross-linking reaction to form the cell aggregate (300). ) is also possible to form.

Using the same principle and method, a hollow sphere-shaped hydrogel structure can be formed through an immediate binding reaction for protein-based hydrogels using a cross-linking agent.

At this time, it is very important to control the porosity of the hollow spherical hydrogel formed, and it must have a porous structure above a certain level, but form a stable structure above a certain level in order to effectively prevent physical contact between cell aggregates. Must be able to.

In particular, in order for the biological agent secreted or produced in the cell aggregate to easily move outside the hydrogel structure, it must have a pore shape at least the size of the biological agent and an appropriate level of porous structure. However, if the thickness of the hydrogel structure is too thick or the porosity is too low, When the porosity is low, biological agents secreted or produced from cell aggregates accumulate only inside the hydrogel structure and are difficult to discharge smoothly to the outside of the hydrogel structure.

Therefore, in order to secure the mechanical strength of the hydrogel structure formed by the immediate bonding reaction and maintain an appropriate level of porosity, Fe ³⁺ , Al ³⁺ , Cr ³⁺ , Cu ²⁺ , Ba ²⁺ , Sr ²⁺ , In the case of alginate used for ionic crosslinking using divalent or trivalent metal cations such as Ca ²⁺ , it is preferably used in a concentration range of 0.1 wt% or more and less than 2 wt%.

Below, the specific actions and effects of the present invention will be explained through specific examples of the present invention. However, this is presented as a preferred example of the present invention, and the scope of the present invention is not limited by the examples.

[Example 1]

Through cell culture, ⁶

Dissolve gelatin powder (cold water fish, or porcine skin) in distilled water to prepare 1ml of 10wt% gelatin solution, then CaCl ₂ (Sigma Aldrich) was further mixed into the gelatin solution to a concentration of 100mM to obtain 5wt% gelatin and A bioink containing cells (first mixture) was prepared.

[Example 2]

After preparing alginate solutions of various concentrations and filling them in an open-top container, the container was placed under a volumetric precision dispenser, and the bioink (first mixture) prepared in Example 1 was added to a syringe for the volumetric precision dispenser. After transferring, the remaining bubbles inside the syringe and volumetric precision dispenser were removed.

It was discharged using a dispenser with a nozzle with an internal diameter of 200 ㎛, and at this time, the position was adjusted so that the bioink (first mixture) discharged through the drive control of the x-y-z stage linked to the dispenser could be maintained at a certain distance without overlapping each other. did.

The calcium ions contained in the discharged bio-ink are ion-cured in-situ as they come in contact with the alginate solution filling the container, and the bio-ink, which is the first mixture, forms a hydrogel structure in the form of a hollow sphere. .

At this time, changes in the thickness and porosity of the wall of the hydrogel structure generated by changing the concentration of alginate discharged from the bioink (first mixture) were observed, and the results are shown in Figure 3 (a). It is summarized in (d) and Figure 4.

(a) to (d) of Figure 3 shows the hydrogel structure formed through in-situ ionic crosslinking reaction and located inside when the concentration of the alginate solution was changed to 0.1, 0.5, 1.0, and 2.0 w/v%, respectively. From a photograph observing the cell aggregate, it was confirmed that when the same concentration of calcium, an ionic cross-linking agent, was used, the wall thickness of the hydrogel structure decreased as the concentration of alginate increased.

Figure 4 shows the results of measuring the porosity of the wall of the hydrogel structure according to the change in alginate concentration through a plurality of repeated experiments including the photographs observed in Figure 3. To measure the porosity of the wall forming the hydrogel structure, first measure the weight (Wi) and volume (Vi) of the dried hydrogel structure, and then soak the dried hydrogel structure in isopropanol (density ρ). After soaking for 15 minutes, the weight (Wf) was measured and calculated using equation (1) below.

[((Wf-Wi)ⅹρ / Vi]ⅹ100 Equation (1)

According to the results of FIG. 4, it can be seen that the porosity of the hydrogel structure decreases as the concentration of alginate increases at a constant cross-linker concentration, similar to what was previously confirmed in the photograph of FIG. 3.

[Example 3]

Cell aggregates were cultured using the alginate hydrogel hollow sphere structures having various porosity prepared in Example 2.

Depending on the change in porosity of the alginate hydrogel hollow sphere structure, can cell secretion factors (secretome) secreted or produced from cultured cell aggregates be smoothly discharged to the outside of the microbioreactor (i.e., outside of the hydrogel structure)? To confirm this, hydrogel structures with various porosity were manufactured by changing the alginate concentration.

After placing cell aggregates in hydrogel structures with different porosity, a culture process is performed, samples are collected from the outside of each different microbioreactor, and the number of secretomes contained in the samples is measured. It was measured using a nanoparticle tracking analysis (NTA), ZetaView110, Particle Metrix.

The number of cell secretion factors per cell measured in this way was converted to a relative number by taking the number of cell secretion factors produced by cell aggregates formed of the same type of cells without a hydrogel structure as 100, and the results are shown in Figure 5. It is summarized in .

As confirmed in the results of Figure 5, when the concentration of alginate used was low (0.1 to 1.0 wt%), it was found that the porosity of the wall of the hydrogel structure was maintained high in the range of about 90% or more, and the hydrogel structure The concentration of biological agents was observed to be high outside the microbioreactor, almost regardless of the thickness, and at almost the same level as in the case without the hydrogel structure.

In other words, when the porosity of the hydrogel structure wall was high, cell secretion factors easily penetrated the hydrogel structure wall and were administered at a high concentration outside the microreactor. However, when the concentration of alginate increased to 2.0 wt%, alginate hydro The porosity of the gel structure was significantly reduced to less than 90%, indicating that cell secretion factors produced or secreted by the cell aggregates were hardly discharged to the outside of the hydrogel structure.

On the other hand, when the wall porosity of the hydrogel structure is 99% or more, the physical strength of the hydrogel structure is significantly reduced, and it is difficult to prevent or block the physical aggregation or fusion of cell aggregates during the culture process, as seen in Figure 1. do.

Therefore, it is preferable that the porosity of the wall of the hydrogel structure is 90% or more and less than 99%, and more preferably, it is necessary to maintain a porosity of 92.3 to 95.6%. In particular, when using alginate to form an ion cross-linked hydrogel structure, the alginate concentration is 0.1 wt% or more and less than 2 wt%, more preferably, the alginate concentration is used in the range of 0.1 wt% or more and 1.0 wt% or less. It can be seen that it is desirable to form an ion cross-linked hydrogel structure in a microbioreactor.

The present invention is not limited to the specific embodiments and descriptions described above, and various modifications can be made by anyone skilled in the art without departing from the gist of the invention as claimed in the claims. and such modifications fall within the protection scope of the present invention.

100: Micro bioreactor
200: Hydrogel structure
300: Cell aggregate
400: Biological agents

Claims (15)

It includes a microbioreactor in which cell aggregates are located within a hydrogel structure in the form of a hollow sphere,
The cell aggregates and the hydrogel structure are spaced apart from each other, and a space exists between the cell aggregates and the hydrogel structure,
The cell aggregate located within the hydrogel structure is a single cell aggregate formed by aggregation of individual cells,
The hydrogel structure is an immediate bonding reaction hydrogel ( in-situ forming or rapid forming hydrogel), which is a polysaccharide hydrogel crosslinked by ions,
The polysaccharide hydrogel is at least one selected from alginate, chitosan, pectin, and poly(N-isopropyl acrylamide),
The ion used for crosslinking the polysaccharide hydrogel is at least one metal selected from the group consisting of Fe ³⁺ , Al ³⁺ , Cr ³⁺ , Cu ²⁺ , Ba ²⁺ , Sr ²⁺ and Ca ²⁺ It is a cation,
A biological agent production device, characterized in that the concentration of the polysaccharide hydrogel is 0.5 wt%, and the porosity of the hydrogel structure is 94.0 or more and 94.2% or less.
delete delete According to paragraph 1,
A biological agent production device, wherein the biological agent produced or secreted from a single cell aggregate located within the hydrogel structure is discharged to the outside of the hydrogel structure.
delete According to paragraph 1,
A biological agent production device, characterized in that the biological agent is an extracellular vesicle, a cell secretion factor, an exosome, an antibody, or a viral vector.
According to paragraph 1,
The cell aggregate is a biological agent production device in which a plurality of human-derived cells, animal cells, plant cells, genetically modified cells, or host cells are aggregated.
delete delete delete delete A microbioreactor in which cell aggregates are located inside a hollow sphere-shaped hydrogel structure is cultured in a culture medium,
Biological agents produced or secreted from the cell aggregates located within the hydrogel structure pass through the hydrogel and are discharged to the outside,
The hydrogel structure is an immediate bonding reaction hydrogel ( in-situ forming or rapid forming hydrogel) of polysaccharide hydrogel crosslinked by ions,
The cell aggregate and the hydrogel structure are spaced apart from each other, and a space is formed between the cell aggregate and the hydrogel structure,
The cell aggregate located inside the hydrogel structure is a single cell aggregate formed by aggregation of individual cells,
The polysaccharide hydrogel is at least one selected from alginate, chitosan, pectin, and poly(N-isopropyl acrylamide),
The ion used for crosslinking the polysaccharide hydrogel is at least one metal selected from the group consisting of Fe ³⁺ , Al ³⁺ , Cr ³⁺ , Cu ²⁺ , Ba ²⁺ , Sr ²⁺ and Ca ²⁺ It is a cation,
A method for producing a biological agent, characterized in that the concentration of the polysaccharide hydrogel is 0.5 wt%, and the porosity of the hydrogel structure is 94.0 or more and 94.2% or less.
delete delete According to clause 12,
A method for producing a biological agent, characterized in that the microbioreactor is statically cultured or dynamically cultured in a culture medium.