EP2978838A1

EP2978838A1 - Method and device for producing a cell culture of human or animal cells

Info

Publication number: EP2978838A1
Application number: EP14713437.3A
Authority: EP
Inventors: Günter BERTHOLDT
Original assignee: Xellutec GmbH
Current assignee: Xellutec GmbH
Priority date: 2013-03-25
Filing date: 2014-03-25
Publication date: 2016-02-03
Also published as: DE102013005198A1; DE102013005198B4; US20160024462A1; CN105051182A; WO2014154679A1

Abstract

The invention relates to a method for producing a cell culture (9) of human or animal cells, more particularly mammalian cells, for the purpose of implementing medical or pharmacological assays. In accordance with the invention, the method comprises at least the following steps: inserting a substrate (3) of microbially produced cellulose into a vessel (1); introducing a nutrient solution (2) into the vessel (1); applying human or animal cells (4) to the substrate (3); conditioning of the vessel contents to approximately body temperature; and waiting until the substrate (3) is completely covered by a cell lawn (5), i.e. confluence has been reached. Medical or pharmacological assays can then be carried out on the cell culture (9) thus produced.

Description

description

Method and device for producing a cell culture from human or animal cells

The invention relates to a method for producing a cell culture from human or animal cells according to the preamble of

Patent claim 1, a microtiter plate with a plurality of wells filled with a cell culture, according to the preamble of claim 12, and a use of a cell culture according to the preamble of patent claim 14.

Medical or pharmacological tests that investigate the effect of certain drugs typically target cell cultures

human or animal mammalian cells. The cell culture is exposed to the substances to be tested and the reaction of the cells is examined.

For medical or pharmacological tests, cell cultures are usually used today in which cells are grown on a surface such as a cell surface. cling to the bottom of a cell culture vessel. For this purpose, a suspension of human or animal cells is pipetted into a vessel in which a

Nutrient solution is included. The cells then sediment in the nutrient solution and settle at the bottom of the vessel. There they attach and divide at a rate that is essentially dependent on the growth factors contained in the nutrient solution. After a few days, a so-called cell lawn forms on the bottom of the vessel, completely covering the soil. However, this cell lawn is stable only for a limited time. If the cells remain in the vessel too long - usually after a few days - the cells will lift off the bottom of the vessel and die. It is therefore necessary at regular intervals a so-called passage

in which the cells from the bottom of the vessel by means of an enzyme detached, washed, isolated and then pipetted into another vessel for re-cell division. This process is called passenger and usually done by hand. He is relatively expensive and expensive. In addition, the cells change after each passage, their phenotype and their specific properties. That is, they dedifferentiate and develop from a more differentiated stage with very specific properties, such as: As skin cells or liver cells, too nonspecific and ultimately for

Investigations of unsuitable cells. In principle, cells of a younger passage (daughter generation) are usually less differentiated than cells of an older passage (parent generation). After several passages, the cells contained in the suspension finally have completely different properties than the original starting cells.

As a result, the same medical test, which is performed once on a cell culture of an older and once on a cell culture of a younger generation, can give different results. Therefore, the tests made on the known cell cultures are reproducible only for a very short period of a few days, as the cell culture changes over time.

Another disadvantage of cells that constantly divide is that the cells are in a condition that differs significantly from the situation in a tissue of an adult organism. Cell division in an adult body is a relatively rare event. It usually occurs only after tissue loss and usually goes with one

Inflammatory reaction associated. As a result, one does not investigate the normal state of these cells, but actually an extreme situation. This fact may explain why, so far, test results obtained using such cell cultures are often poorly consistent with clinical findings.

In contrast, since it has not been possible to keep dormant cells alive for a long time, there has been a trend to use especially dividing cells for pharmacological tests and to use them

Shorten observation period. However, this leads to the fact that the Results are even less meaningful, as can easily be explained from the above.

Disclosure of the invention

It is therefore an object of the present invention to provide a method for producing a cell culture from human or animal cells for carrying out medical and pharmacological tests, with which a cell culture can be produced which is stable over an extended period of time, in particular over several weeks. This makes it possible to perform medical or pharmacological tests at intervals of several weeks on the same cell culture. In addition, it is an object of the present invention to provide a long-term stable cell culture produced by the method according to the invention, at which then the desired

medical or pharmacological tests can be performed.

The above objects are achieved according to the invention by the features stated in the independent claims. Further

Embodiments of the invention will become apparent from the dependent claims.

According to the invention, a method for producing a cell culture from human or animal cells, in particular from mammalian cells, for carrying out medical or pharmacological tests is proposed which comprises at least the following steps:

- inserting a substrate of microbially produced cellulose into a vessel;

- filling a nutrient solution into the vessel;

- applying human or animal cells to the substrate;

- tempering the vessel contents to a predetermined temperature,

in particular body temperature;

Wait until the substrate is completely covered with a cell lawn, i. that all cells have touched and stopped dividing (which may take a few days to several weeks, depending on initial concentration and cell type);

Preferably, it is tested on the basis of metabolic parameters whether and when the cells have reached their characteristic state and - Supplying a drug to be tested to the on-substrate cell culture.

After application of the human or animal cells to the microcrystalline cellulose substrate, a cell division and cell division occurs

Growth process in which the substrate is increasingly covered by cells. This process works optimally at body temperature, ie at about 37 ° C. Therefore, during the cell division and growth process, the cell culture is preferably maintained at about 37 ° C. When the cells cover the substrate so tightly that the outer membranes of the cells touch each other completely, the cells stop dividing. After setting the

Division process, the cells need a certain amount of time to change and reach a so-called dormant phenotype. The term "dormant" refers only to the fact that there are no more dramatic changes in shape as in cell division.For biochemical reasons, these cells are very active and produce factors that are typical for their differentiation state microbially produced cellulose stable for at least several weeks.

With regard to the individual process steps of the process according to the invention, it should be emphasized that these do not necessarily have to be carried out in the stated sequence. Thus, for example, the application of human or animal cells to the substrate can also take place before the introduction of a nutrient solution into the vessel. Optionally, the nutrient solution could first be filled into the vessel and then the substrate is inserted first. It is clear to the person skilled in the art that he can determine the sequence of the individual steps to a certain extent as desired.

The cells may be slow to storage or for transport

Room temperature of z. B. 15 ° -30 ° C, in particular 18 ° -25 ° C are cooled.

It is not necessary to freeze the cell culture for storage or transportation as it remains stable at room temperature.

According to a preferred embodiment of the invention, the cell culture is preferably prepared from stem cells or from cells obtained from stem cells, such. Liver, heart or kidney cells, or cells other organs, such as B. the skin. A medical or pharmacological test can thus be carried out on known, well-defined cells. In the case of cells derived from stem cells, these cells may be e.g. Example, by means of a method, as it is known from US 20030161818A1 or US 2003 015 45 06 A1. Here, the stem cells are obtained from human umbilical tissue. The stem cells are located in the so-called substantia gelatinea funiculi umbilicalis, a gelatinous matrix rich in hyaluronic acids and chondroitin sulfates, also called "wharton's jelly", which is extracted by an enzymatic process (eg collagenase).

Alternatively, of course, it is also possible to use those cells which have been taken directly from a human or animal body. This happens e.g. via a biopsy from the bone marrow or from the adipose tissue.

According to one embodiment of the invention, for. B. cells are used, which come directly from affected patients, creating a

personalized therapy becomes possible.

The cells applied to the cellulose substrate are preferably those cells which have not previously been passaged. The cell culture thus arises according to the invention by a unique cell division and growth process of original, so-called. Primary cells such. As stem cells or derived cells that have not gone through a dedifferentiation process. That is, the cells applied to the cellulose substrate are preferably cells having a zero passage number. Optionally, however, it would also be possible to use cells with a passage number of less than or equal to five.

For transport, it is not necessary to freeze the cells as usual; the transport can rather be done at ambient temperature, such. B. between 15 ° C and 30 ° C. The temperature can also be higher or lower.

When the ambient temperature is within the desired range, e.g. B. between 15 ° C and 30 ° C, it is sufficient to allow the cell culture to cool easily (without using a cooling device). The cell culture could also be active, ie by means of a cooling device to be cooled down to the desired temperature.

According to a preferred embodiment of the invention, the vessel is provided with a hermetic seal, e.g. a foil or a lid provided. In particular, the hermetic seal is intended to prevent the entry or exit of media such as liquid or gas from or into the vessel. The sealing of the vessel can in principle be carried out before or after cooling, but is preferably before

carried out.

According to a preferred embodiment of the invention, the vessel is sealed with a foil, e.g. is welded or glued to the vessel. Alternatively or additionally, a lid could be provided which closes off the vessel.

The vessel with the cell culture therein can finally be packaged, then z. B. to a laboratory or a customer, who then performs the medical or pharmacological tests on the cell culture. The packaging may comprise, for example, a box and optionally insulating material. The packaging may also include a coolant.

In order to protect the cell culture after their preparation, in particular for subsequent transport by mail, or generally against mechanical stress and shock, it is proposed to increase the viscosity of the nutrient solution substance or a substance with a higher viscosity than that

Add nutrient solution into the vessel. According to the first alternative, the nutrient solution can be mixed, for example, with methyl cellulose, polyethylene glycol or another viscosity-increasing substance. According to the second alternative, a higher-viscosity substance, such. B. a gel-like substance are introduced into the vessel. But it could also be a solid, such. B. a body of microcrystalline cellulose can be placed on the cell culture. As a result, the cell culture is mechanically held on the bottom of the vessel and can only be difficult to tilt to the side or float. According to a preferred embodiment of the invention, the cells of the cell culture are not passaged, ie the cell lawn is preferably not detached and the cells are not separated as in the prior art and then applied again to another substrate for re-cell division. Rather, the cell culture is preferably prepared by a unique cell division and growth process from original cells, ie, cells that have not previously been passaged.

The cell culture is preferably packaged in the same vessel in which it has been cultured and optionally sent to a recipient. It does not have to be transferred to another vessel.

According to a preferred embodiment of the invention, the above method is performed on a microtiter plate having a plurality of wells. In each of the wells, a substrate of microbially produced cellulose is inserted, filled in a nutrient solution and applied to the substrate or human or animal cells. Thereafter, the cells grow and divide until at least partially covering the substrate. Finally, each cell of the microtiter plate contains a part of the same cell culture. The individual subcultures can then be used to perform the same or different medical or pharmacological tests.

The storage or transport of the cell culture is preferably carried out at

Ambient temperature. A freezing of the cell culture, as usual, is not required.

The entire microtiter plate is preferably sealed with a hermetic

Provided closure as described above. It can finally be packed and sent.

The invention also relates to a microtiter plate having a plurality of wells, each filled with a substrate made of microbial cellulose which is populated by a cell culture. The microtiter plate is located

preferably at about ambient temperature and is neither refrigerated nor heated. It could also be stored at temperatures below room temperature, eg. B. be cooled to 5 ° C to 10 ° C.

According to a preferred embodiment of the invention, a substance which is arranged on the cell culture and has a higher viscosity than the nutrient solution is contained in the individual wells of the microtiter plate.

As a result, the cell culture is held together with the substrate mechanically at the bottom of the vessel and can only be difficult to tilt to the side or float.

Moreover, the invention also relates to a use of a cell culture from human or animal cells, in particular mammalian cells, which has been prepared according to one of the methods described above, for carrying out medical or pharmacological tests.

The invention will now be described by way of example with reference to the accompanying drawings. Show it:

Fig. 1 a - 1 h different states of a method for producing a

Cell culture from human or animal cells according to an embodiment of the invention; and

Fig. 2 is a microtiter plate with several wells in which

in each case a cell culture produced according to the method according to the invention is located.

Figures 1 a-1 h show various states of a method for

Producing a cell culture 9 from human or animal cells. The cell culture 9 can later be used to perform medical or pharmacological tests.

To produce the cell culture 9, a vessel 1 with a first

Nutrient solution 2 filled (Fig. 1 a). As nutrient solution 2, for example, the nutrient medium of Schramm and Hestrin described in Biochemical Journal 58 of 1954, pages 345-352 can be used. The nutrient solution may, for example, consist of 20 g of glucose, 5 g of yeast extract, 5 g of bactopeptone, 2.7 g of sodium phosphate and 1.15 g Citric acid monohydrate and 0.5 g of magnesium sulfate heptahydrate in one liter of water. Alternatively, other nutrient solutions known in the art may also be used.

As a next step, a substrate 3 made of microbial cellulose, which may for example have the form of a platelet, is then placed in the vessel 1 (FIG. 1 b). The two steps described above can also be performed in reverse order. A substrate 3 of microcrystalline cellulose can be produced, for example, by means of a method as known from DE 10 2008 056 413.3 or WO 2010 052 019 A2. Other manufacturing processes for manufacturing

microcrystalline cellulose are well known in the art.

In Fig. 1 c, a suspension of human or animal cells 4 is finally introduced into the vessel 1. The applied to the substrate 3 cells can, for. B. stem cells or cells derived from stem cells, such as. As liver, heart or kidney cells, or cells of other organs, such as. B. the skin. The cells 4 can z. Example, be prepared by a method, as it is known from US 02013025983A1, US 6410320B1 or US 7534607B1. Alternatively, of course, it is also possible to use those cells 4 which have been taken directly from a human or animal body.

The cells 4 applied to the cellulose substrate are preferably those cells which have not previously been passaged. The cell culture 9 thus arises from a unique cell division and growth process of original cells that have not undergone a dedifferentiation process. That is, the cells 4 applied to the cellulose substrate 3 are preferably cells having a passage number equal to zero.

The cells 4 then sediment in the nutrient solution 2 and settle on the surface of the cellulose substrate 3. At temperatures around 37 ° C then takes place a division and growth process, which forms on the substrate 3 within a few days, a so-called cell lawn 5, which completely covers the substrate 3, as shown in Fig. 1 d. Once the cell lawn 5 has reached the desired size and thickness, the cell division and growth process stops. The vessel 1, including its contents, is then cooled to a lower temperature. According to a preferred embodiment of the invention, the vessel is allowed to cool to room temperature, e.g. B. to about 18 ° C to 25 ° C. It has been found that the cell culture 9 at these temperatures over several weeks, z. B. remains stable over three, four or even more weeks and in particular the cells do not die. Depending on the type of cells, tests can now be carried out on the cells, for example addition of substances for the formation of new blood vessels on endothelial cells, the effect of possible toxic substances on liver cells, or the influence of the beat frequency of cardiac muscle cells.

Optionally, in a further step 1e, an agent 10 increasing the viscosity of the nutrient solution 2, such as e.g. Methyl cellulose or polyethylene glycol (PEG) are added. This results after the addition of the agent 10

Medium is marked in Fig. 1f with the reference numeral 1 1.

Instead of the agent 10 or in addition, a substance with a higher viscosity than the nutrient solution or a solid could also be applied to the cell culture 9. So z. B. another body of microcrystalline

Cellulose can be placed on the cell culture 9. This can be prevented that the substrate 3 lifts off together with the cell culture 9 from the bottom of the vessel 1 and z. B. tilts or floats to the side. In a further step 1f, the vessel 1 can still be hermetically sealed

Seal 6 are sealed to prevent the escape or penetration of medium. The hermetic seal 6 may be, for example, a foil which may be e.g. can be welded or glued to the vessel 1. Optionally, of course, a lid with a seal (not shown) could be provided.

To send the cell culture 9 including the vessel 1, the vessel 1 is finally packed in step 1 g. In the illustrated embodiment, the vessel 1 is packed in a box 12 and additionally protected by a filler 13 against mechanical shocks and damage. According to the invention, the cell culture 9 is preferably shipped in the same vessel 1 in which it has also been cultivated.

From the individual process steps 1 a to 1 g it is clear that it is not necessary to pass the cells of the cell culture 9, i. the cell lawn 5 is not detached from the substrate 3 and the cells are not separated and then reapplied to another substrate for re-cell division. The cell culture 9 is rather produced in a single growth and cell division process.

Finally, FIG. 1 h also shows how a substance 14 to be tested is applied to the cell culture 9, while the cell culture 9 is still located on the substrate 3. The reaction of the cells to the substance is then diagnosed.

FIG. 2 shows a microtiter plate 8 with a plurality of wells 7, each serving as a vessel 1 according to FIGS. 1 a to 1 g. In each of the wells 7 is a substrate 3 of microcrystalline cellulose, which is populated by a cell culture 9. The preparation of the cell cultures 9 can be produced simultaneously in accordance with the method steps shown in FIGS. 1a to 1d.

After the individual cell cultures 9 have reached the desired size, the microtiter plate 8 is cooled to room temperature and z. B. closed by means of a film 6, as shown in Fig. 1f. In each of the wells 7 is then part of the same cell culture.

If the microtiter plate 8 is to be shipped, it is packed in a further step according to FIG. 1 g. The customer thus receives one

Microtiter plate with a ready-for-many weeks stable cell culture 9, where he can perform the desired medical or pharmacological tests.

Claims

claims

1 . Method for producing a cell culture (9) from human or

animal cells, in particular mammalian cells, for carrying out medical or pharmacological tests, comprising the following steps:

Inserting a substrate (3) of microbially produced cellulose into a vessel (1);

Filling a nutrient solution (2) into the vessel (1);

Applying human or animal cells (4) to the substrate (3);

Tempering the vessel contents to a predetermined temperature, preferably to about body temperature; and

Wait until the substrate (3) is completely covered by a cell lawn (5), the cells no longer divide and have assumed a quiescent phenotype.

2. The method according to claim 1, characterized in that the cell culture (9) is cooled after its preparation to about room temperature.

3. The method according to claim 1 or 2, characterized in that the

Cell culture (9) before performing a medical or

pharmacological tests is not frozen.

4. The method according to any one of the preceding claims, characterized

in that the cells (4) applied to the substrate (3) are stem cells or cells derived from stem cells.

5. The method according to any one of the preceding claims, characterized

in that the cells (4) applied to the substrate (3) have not previously been passaged or have a passage number of less than or equal to five and in particular less than or equal to two.

6. The method according to any one of the preceding claims, characterized

in that the vessel (1) is provided with a hermetic seal after the production of the cell culture (9) and before the medical or pharmacological test has been carried out.

7. The method according to any one of the preceding claims, characterized

characterized in that the cells (4) are not passaged, that is, the cell lawn (5) is not detached from the substrate (3), and the cells (4) are not singulated and then re-applied to a substrate (3)

Cell division applied.

8. The method according to any one of the preceding claims, characterized

characterized in that the cell culture (9) in the same vessel (1), in which it was grown, is packaged.

9. The method according to any one of the preceding claims, characterized

in that the method is carried out simultaneously on a plurality of vessels (1, 7).

10. The method according to claim 9, characterized in that the vessels

Wells (7) of a microtiter plate (8).

1 1. Method according to one of the preceding claims, characterized

in that a substance (10) which increases the viscosity of the nutrient solution (2), or a substance with a higher viscosity than the nutrient solution (2), is additionally introduced into the vessel (1, 7).

12. A microtiter plate (8) with a plurality of wells (7), each filled with a microbial cellulose substrate (3) populated by a cell culture (9), characterized in that the cell culture (9) comprises cells that have not been previously passaged, and that the individual wells (7) are hermetically sealed.

13. Microtiter plate (8) according to claim 12, characterized in that it additionally contains a substance (10) which increases the viscosity of the nutrient solution (2), or a substance with a higher viscosity than the nutrient solution (2) which is used on the cell culture ( 9) is arranged.

Use of a cell culture (9) from human or animal cells (4), in particular mammalian cells, which has been produced in accordance with a method as claimed above, for carrying out medical or

pharmacological tests.