DD159181A1 - METHOD FOR CULTIVATING CELLS - Google Patents

Abstract

The invention relates to a method for cultivating cells, in particular embryonic mouse fibroblasts. Field of application is cell breeding, in the broader sense human and veterinary medicine as well as drug discovery. The aim of the invention is to improve the detachment and separation of cells from cell cultures and tissues as well as in the passage of cells. The task of developing a cultivation method with the aid of an enzyme of high activity was achieved by allowing the enzyme thermitase to be applied to cell cultures and tissue, in particular embryonic mouse fibroblasts, for the production of live cell suspensions.

Description

The invention relates to a method for culturing cells, in particular embryonic mouse fibroblast cultures, using the Bnzyms thermitase _ö With the aid of the inventive method, the separation and isolation of cells from cell cultures succeeds and tissues with high efficiency - even without the addition of Complexing agents - as a result of the relatively low enzyme concentration and the short exposure time biologically important structures of the cell membrane are largely spared ©

The invention is of relevance to the pharmaceutical, human and veterinary medicine, and more particularly to drug discovery for the testing of drugs for their pharmacological action (mechanism of action, structure-activity relationships, receptor studies) and their toxicity in experimental and practical medicine Cell breeding increasingly widespread application in virology, u »a« for vaccine production, in cancer research as a method for tumor diagnostics and cytostatic drug efficacy tests in tumor therapy, in mutagenicity research, cytodiagnostics (for the detection of chromosomal abnormalities, z _e B · in the Amniozetese) and in the immunology OU hall, in the Zeil and tissue breeding with animals, VSB Gustav Piseher «publishing house, Jena 1976) *

Characteristic of the known solutions

It is known to use enzymes in cell culture. Thus, for the cultivation of animal cells in a variety of methods and manipulations, such as cell detachment and »isolation, production of cell suspensions or subcultivation and passaging proteolytic enzymes used to avoid cell damage, the acting protease concentration and the exposure time are kept as low as possible (Corziell , Lo C · in Methods Enzymology £ 8 ^ 29, Jakoby and J., Paetan (Hrsg *) (1979). In most methods, trypsin is used with and without the addition of ethylenediamine tetraacetate (EDTA) (Puck , Τβί J «Exp« Med · KHg, 615 (1962) Mc Keehan, W · L «: Cell, Biol _β Into Rep, I ^ 335 (1977) Weinstein, D .: Εχρ _β Cell« Res ^ j 234 (1966) Seglen, P. * s Exp. Cell. Res * 82 ^ 391 (1973) Steinberg, Mo S. j. Exp. Cell · Res. £ 0 ^ 257 (1963) · The enzyme concentration is between 0.1 mg / ml (Basher, M ₀ Μ · in: Methods in Enzymol. _< §8 _X 119, W, B. Jakoby and J. H. Pastan (Hrsg *) (1979)) u 2.5 mg / ml (Mc Meer, J. Α and W. Η _β J. Douglas, ibid. ^ 8 ^ 132) at an E inv / irkungszeit 2-15 minutes and an incubation temperature between 6 ⁰ C and 37 ⁰ C (Mc Keehan, W. L ,, Cell "Biol. Int. Rep. I ^ 335 (1977), Basher, M.M., in: Meth. In Enzymol. £ 8 ^ 119 (1979)) "Lifting trypsin under similar conditions were other hydrolytic enzymes, such as pronase, collagenase, hyaluronidase _? Elastase and Papain (Wisemann, L "L. and W _e R" Hammond, J "Expl., Zool. V) J ^ 429 (1976)). Thermitase, the protease major component of the culture filtrate of Thermoactinomyces vulgaris, has not yet been used in cell culture *. It has a proteolytic activity comparable to casein, such as trypsin, without possessing a pronounced specificity for particular cleavage sites (Behnke, U ₀ , R _e Kleine, M "Ludewig

and H ₀ Euttloff, Acta biol «med» germ «^ Za. 1205 (1973); Kleine, H. and U _e Rothe, Acta biol * med. Germ. .36, K 27 - K 33 (1977)).

The object of the invention is to improve the detachment and separation of cells from cell cultures and tissues as well as the passage of cells in the field of cell culture, by shortening the time of action of the enzyme solution and decreasing the enzyme concentration used, so that the cells in the primary and subcultures show a high rate of attachment, a normal, regular cell shape and a good and rapid increase »

Pariegung_jäeg_Wesens of the invention

The invention has for its object to develop a method for cell cultivation, which starts from an enzyme with high efficiency and which can therefore be used in low concentration in the detachment and separation of cells from cell cultures and tissues. The object has been achieved by using * thermitase as enzyme in the culture of embryonic mouse fibroblasts. [0048] For detachment and separation of cells from cell cultures, a thermitase solution in the form of a thin liquid film is allowed to act on the culture for a short time (a few seconds) and then suspended the cells in a common culture medium

A comparison of the efficacy of thermitase and trypsin shows that thermitase is similarly effective at lower enzyme concentrations and without addition of EDTA for cell detachment and isolation. A concentration of 0.05 mg / ml thermitase corresponds to the efficacy of a

Trypsin solution of 1.25 mg / ml with the addition of 4 ml of EDTA "In" both cases, no dead cells are detectable with trypan blue "The subcultures after ter- metitase treatment show no abnormalities compared to those after trypsin treatment" The growth rate of the cells in all subcultures after thermitase dissolution is similar to that after treatment with trypsin, the cell form is regular * ¹

The detachment and separation of cells from biological material, z » ^; B »Mice embryos, for the production of cell suspensions, succeed by the action of thermitase in a fraction of the concentration of trypsin« The assessment of cell detachment from the substrate or the tissue »cell separation, cell attachment rate, the cell shape and the determination of the percentage of dead cells after enzyme action by light microscopy, optionally with the aid of a suitable counting chamber, the quantitative evaluation for the detection of cell proliferation in the cultures with an electronic particle counting devices

The invention will be explained in more detail with reference to embodiments "^;

Example 1 ....... ....

Preparation_vpn cell suspensions from monolayer cultures of

Assessment of their quality by *

Cell Removal Rate Cell Separation (Zeilhaufenbildung, % of individual parts)

- percentage of dead cells

- cell morphology

The cultivation of the fresh mouse embryos after treatment with trypsin (2.5 mg / ml trypsin at 37 ⁰ C for 25 to 30 minutes) obtained primary cells is carried out in 25 ml Müller bottles in the form of monolayer cultures at 37 ⁰ C in 5 ml of the culture medium Minimal Essential Medium (MBM, Staatl, Institute for Immuno-Preparations and Mahrmedien, Berlin) with the addition of 0.014 % penicillin, 0.01 % streptomycin, 4.2 mM HaHCOo and 10% bovine serum (RS).

Enzymtestlösungern

Wii 1 IMWiIiBtHi im hu nium n * 11 m ι r in ti!

} The enzymes trypsin (for cell cultivation, Leithold, Kleinmachnov and Thermitase (purified according to the instructions of Frömmel, C, G. Hausdorf, WE Hohne, U, Behnke and H _e Ruttloff, Acta biol «med germ, ^ Ls. 1193 ( 1978)) or _e the complexing agent disodium dihydrogenethylenediaminetetraacetate (VEB Berlin-Chemie) are each in appropriate concentration (Table) in isotonic buffer (PBS, State Institute for Immuno-Preparations and Efährmadien, Berlin) with the addition of the antibiotics penicillin (0.014 %) and streptomycin ( 0.01 %) and then sterile filtered (miliporfilter, diameter 0.45 / tm)

j detachment and separation of cells and production of

After removal (decanting) of the culture medium from the solid cell lawn, it is wetted with a small amount of the appropriate enzyme solution (pour off excess liquid immediately). This enzyme liquid film is allowed to act for a short time (a few seconds to a maximum of 2 minutes, Table) and mechanically suspends the cells thus dissolved mechanically in the culture medium MEM #

The evaluation of these cell suspensions is carried out by light microscopy (speed of cell detachment, cell morphology) or, using a Fuohs-Rosenthai cell counting cell (cell separation, cell clustering) and the dye 2rypan blue (0.3 % to 0.5 % i detection of dead cells).

Table: Influence of thermitase © or · ¹ trypsin on cell detachment and separation

Concentration cell detachment

mg / ml

Cell Separation 0- Number of cell clusters (from 3 and more cells) per 100 individual parts

Size of cell clusters dead cell form cells of the adherent cells

Thermitase 0 _s 1

0.05

0.02

0.01

within seconds completely

not determined

n · ¹ none

to-

 weisbar

in less than a minute, completely

8 to 9

3 to 4 cells

within

of minutes. , depending on the mechanism "suspension"

XL · · W

not available

b "

regular

elongated

fusiform

deleted

trypsin

 2.5

5 minutes and

long in to many, large 32 to 34 to 20 cell heaps, un. , , Cells completely '

Thermitase and 4 EDTA

0,025

within seconds, completely and i g

1 to 2

from max. 3 single cells regular

elongated spindle-shaped

Trypsin and

mM SDTA 1.25

within seconds, completely

2 to 3

3 to cells

tf

n. b · = not determined

This comparison with the standard enzyme mixture of 1.25 mg / ml trypsin and 4 mM EDTA shows that 0.05 mg / ml thermitase is suitable for the detachment and separation of cells and the preparation of cell suspensions of embryonic leukem fibroblasts without any further additives Eate of cell separation after thermitase treatment interferes with further manipulations - such as cell counting with an electronic particle counter and subcultures with normal growth rate -

Not"

Growth Behavior at One Week Cultivation Determination of the Growth Curves of the 1 _e Subculture of .embryonic. Mouse fibroblast en ...

Zellkultivationsbedingungen; Preparation of enzyme

solutions and the cell suspension - see Example 1

From a primary culture of mouse embryo fibroblasts (cultured for 4 days in 80 ml MEM with the addition of 10 % Einderserum, cell seed 3 χ 10 cells) is prepared according to the procedure for the preparation of cell suspensions with the enzyme solutions

a) 1 j 25 mg / ml PBS trypsin and 4 ml EDTA

b) 0.05 mg / ml PBS thermitase;

the first subculture produced * -. Cell dosage 160 000 cells / 5 ml MEM, d _e ho per culture flask + 10 % RS

With the help of the electronic particle counting device TUE ZG2 (VEB Transformatoren- and Böntgenwerk, Dresden), the cells floating in the culture medium and the cells attached to the substrate (after action of a corresponding peeling acting enzyme solution) after β hours, 24 hours and 2, 4 and ? Quantitatively recorded days ",

Growth curves of the subculture of embryonic mouse fibroblasts after treatment with 0.05 mg / ml thermitase ^ * ~ - ~) compared with the standard solution 1.25 mg / ml trypsin and 4 ml EDTA in comparison ')

Six hours after the enzyme treatment, about half of the seeded cells were attached to the bottle base with both enzyme solutions (for the most part already spread in a spindle shape); the other half of the cells are still floating in the medium * Thus, the cell attachment speed is comparable in both cases. "After 1 x day of culture, about three-quarters of all cells have adhered and spread with both cell detachment = subcultivation methods." At this time and also on the following days Cell morphology does not show any significant differences (however, the cells adhere after treatment with ether, but strengthened in the form of aggregates, which, however, dissolve again after 1 day of culture and do not adversely affect the growth rate) (FIG. 1).

Subculture of mouse embryo fibroblast cultures

By means of a thermitase solution of O ₅ 05 mg / ml, subcultivation of embryonic mouse fibroblasts is possible under the conditions and procedures set forth in Examples 1 and 2. In the present example 4 subcultures are applied "every 3 days in a sequential manner, the spent culture medium _s decanted off the cells by means of said enzyme solutions from the cell monolayer detached from the base, resuspended in MEM with 10% bovine serum and the Zeil-

contents of a Koux culture bottle distributed to 2 or 3 others «

Preparation of a live cell suspension and primary culture from fresh mouse embryos

Approximately 15 days female mice were killed by cervical dislocation, bled through a neck incision and dissected out the embryos under aseptic conditions, freed of the fruit sack skin, shredded with a pair of scissors and washed with medium MM.

10 to 15 pre-shredded and washed mouse embryos are then treated with about 50 ml of a Bnzynilösung: Variant a) 0.05 mg / ml Thermitase in PBS variant b) 2.5 mg / ml trypsin in PBS as a comparison. ... enzyme .... . .:

Tissue digestion conditions: 20 to 25 minutes exposure time:

- time of the o # go- enzyme solutions at 37 ⁰ C and with stirring *

The cell-containing digestion solution is separated from the undissolved residue by filtration through a double mullage and centrifuged this supernatant for sedimentation of the cells at 1500 rpm for 10 minutes (laboratory bench centrifuge T 13 "H", Janetzki KG, DDE) * The cell pellet is suspended in 20 ml of MERI , homogenized and alig; oter part removed for electronic particle counting ....: ... '..

The primary culture is cultivated from these cells in Eous culture flasks in 80 ml of MEIiI with the addition of 10 % .Iv.serum at a cell seed of 30 x 10 cells per flask

Starting from approximately equal starting amounts of mouse embryos, the cell yields are

revealing variant

a) 0.05 mg / ml Thermitase 135 χ 10 cells

b) 2.5 mg / ml trypsin 72 χ 10 ⁶ cells

In order to test the survival and growth capacity of the cells treated in this way, they are cultivated and the number of adherent, spread-out fibroblasts is determined (primary culture). ♦ In both cases, after three days of cultivation, about 45 % of the seeded cells (the starting cell suspension contains pibroblasts also a relatively large proportion of erythrocytes) attached to the Glasunterläge' and spread typically fibroblastoid

Cell seed 30 χ 10 cells, per bottle

Number of adherent cells after three days of cultivation

Variant a) 0,05.mg / ml thermitase 13.6 χ 10 cell variant b) 2.5 mg / ml trypsin 10 ° χ 13.7 cells

Claims (3)

Invention claim. 1 * Method for the cultivation of cells, characterized in that the enzyme thermitase acted on cell cultures and tissue for the production of cell suspensions on cell cultures and tissue 2e method according to item 1, characterized in that according to embryonic mouse fibroblast cultures. Removing the culture medium consisting of minimum essential medium (MM) with addition of 0.014 $ penicillin, 0.01% streptomycin, 4 _S 2 ml UaHCO ₃ and 10 la bovine serum, for 0.5 to 2 minutes at 25 to 40 ⁰ C, Preferably at about 37 ⁰ C, a Shermitaselb'sung from 0.02 mg / ml to 0.1 mg / ml, preferably 0.05 mg / inl, and acts after. Remove the enzyme solution and mechanically suspend the cells in MM Method according to item 1, characterized in that the separation and separation of cells from biological material, in particular from mouse embryos, for the production of live cell suspensions by the action of thermitase in concentrations of 0.02 mg / ml to 0.1 mg / ml, preferably from 0.05 mg / ml, at temperatures of 25 to 40 ⁰ C, preferably at about 37 ⁰ C, is made «. For this 1 S? I? @ Drawing