DD225440B1 - CELL MEDIUM BREED - Google Patents

Description

Field of application of the invention

The invention relates to a cell culture medium containing in addition to conventional components a product freshly slaughtered from brain cattle, whereby the proliferation of cells of mesodermal origin, especially human amniotic fluid cells - especially in small cell seed - is promoted in vitro persistently. Thus, it is possible by the invention, in the context of prenatal diagnosis human amniotic fluid cells, which are incurred at the time of their Abnahmemöglichkeit only in very small quantities, in a sufficient for the routine diagnostic examination extent or reasonable time without major laboratory effort and minimizing the risk of Multiply mycoplasma contamination by frequent medium changes in vitro.

The invention is preferably suitable for human medicine - in particular for human genetics. It is also of interest for all biological fields of work, where human or animal cells of comparable species are to be propagated under analogous conditions in vitro for propagation.

Characteristic of the known technical solutions

It is known that very small amounts of animal and human cells, such as those found in amniotic fluid samples in the context of prenatal diagnostics, can be propagated in vitro by conventional cell culture media to which up to 20% fetal calf serum has been added as a growth-promoting component. In certain cases, where the time factor for achieving a suitable or required for the re-use of the cells - such as. For example, for prenatal diagnostic examinations, a further role is played by additional cell growth factors, in particular additions of "fibroblast growth factor" in a largely purified form (1 / xg / ml) (PORRECO, BRADSHAW, SARKAR and JONES, Obstetrics and Gynecology 55, 55 (1980)) or in pure form (100 ng / ml) (GOSPODAROVICZ, MORAN and OWASHI, J. Clin. Endocrinol.

Metab. 44, 651 (1977)) have been found to be generally successful, but here, as in the latter case, the cell seeds intended for propagation are relatively high (about 3000 cells per petri dish / 6 cm diameter /), and furthermore cell culturing is involved in these cases the technical disadvantage of daily or very often required medium renewal.

It is further known that pure "fibroblast growth factor" for unfolding its full cell growth promoting activity on primary cultures and early passages of human diploid cells (fibroblasts), unlike transformed cells, relies on interaction with other biofactors, which is usually partial The addition of serum to the cell culture medium (GOSPODAROWICZ and MORAN, Annual Rev. Biochem., 45, 531 (1976)) thus also explains the multiple medium changes required in the above cases (addition of consumed active substance reserves), with the frequency - apart from the technical aspects Disadvantage - in the case of cultivating human amniotic fluid cells an infection biological risk increases in a very special way, which consists in the fact that a mycoplasma infection of the amniotic fluid cells possibly connected with the renewal of the medium can lead to chromosomal changes of the same - and thus to d may result in diagnostic and diagnostic errors (SCHNEIDER, STANBRIDGE, EPSTEIN, GOLBUS, ABBO-HALSBASCH and RODGERS, Science 184, 477 (1974)).

Likewise with the aim of reducing the risk of mycoplasma contamination - here, however, preferably in the causal relationship with higher serum concentrations (15 to 30%) seen in the medium - successful investigation of a substantial substitution of fetal calf serum in the specific case of the proliferation of human amniotic fluid cells in vitro in the medium by pure "fibroblast growth factor" in conjunction with 9 other biofactors (mainly hormones) has become known, but also here a serum addition (fetal calf serum) of 4% to the medium can not be undershot (CHANG, JONES and MASUI, Proc. Nat. Acad., USA 79, 4795 (1982)). However, even in this case, more frequent medium renewals during cell cultivation can not be dispensed with. NORTHOFF (DE-OS 3,138,597) discloses a method for accelerating the growth of amniotic cell cultures or of human fibroblasts or fibroblast-like cells described in which the stability of pure "fibroblast growth factor" - as well as its solubility - is judged to be critical and a better effect of admixing a

lymphokine-rich supernatant of concavalin A-stimulated lymphocytes is attributed to conventional cell culture medium.

However, the stated range of variation for optimally effective additives (2 to 20% by volume) argues for batch-dependent quality fluctuations, which are a disadvantage for the methodical standardization of cell cultivation. From the same point of view, the limited shelf life (3 months, 4 to 6 ° C) of the admixture also appears problematic.

Object of the invention

The object of the invention is to provide a cell culture medium which, due to its novel composition, is capable of producing cytogenetic small amounts of cells of mesodermal origin, in particular human amniotic fluid cells, as obtained in prenatal diagnostics in amniotic fluid samples or routine biochemical studies within a reasonable period of time (14 or 28 days) with a suitable cell yield and while preserving essential biochemical or cytological parameters in vitro to increase their reproduction. At the same time, the use of this medium is intended to achieve a shortfall of the otherwise normally required frequency of the medium change, which is disadvantageous for various reasons, during cell cultivation.

Explanation of the essence of the invention

The invention has for its object to provide a cell culture medium with particular advantages for the in vitro propagation of cells of mesodermal origin, in particular human amniotic fluid cells in the context of prenatal diagnosis, which can be the conventional cell culture technique in this respect improve.

The object was achieved in that slaughtered cattle were freshly slaughtered from fresh, mechanically cleaned and frosted brains immediately after removal, in accordance with that of GOSPODAROWICZ, BIALECKI and GREENBURG (J.

Biol. Chem. 253, 3736 (1978)) described method by thawing, homogenization and stepwise ammonium sulfate precipitation and by lyophilization of the biological activity-containing precipitation step, a protein preparation was obtained, in addition to "fibroblast growth factor" activity other unspecified components which support the cell growth-promoting potency of "fibroblast growth factor" in the serum-containing cell culture medium in the sense of a lasting efficacy.

This brain preparation is prepared as a ready-to-use admixture with conventional Zellzuchtmsdien.

It is ensured that the entire preparation course proceeds under germ-free conditions and sterile working conditions are maintained even when preparing the admixture. In the preparation of the cell culture media to be supplemented with the preparation and in the admixture of the brain dry preparation, the usual sterilization methods or sterile conditions in cell cultivation must be observed.

The significance of the invention results from the fact that the promotion of the cell growth-stimulating interaction between "fibroblast growth factor" and serum factors, which originates from natural accompanying components of the "fibroblast growth factor", which are present in the brain preparation described above, continues in the manner according to the invention It is used by mixing this brain preparation (dry preparation) in an optimal concentration (100 / ig / ml) to a conventional cell culture medium (eg EAGLE cell culture medium / MEM /) containing 10% (v / v) fetal calf serum, a novel composite Cell culture medium is created, with the one of the requirements of prenatal cytogenetic and biochemical diagnostics in an improved way fair increasing human amniotic fluid cells is made possible, especially the otherwise required frequency of adverse medium renewal can be exceeded.

The invention will be explained with reference to the following embodiments:

Example 1: Obtaining the cell replication-promoting brain preparation

Following the procedure described by GOSPODAROWICZ, BIALECKI and GREENBURG (J. Biol. Chem. 253, 3736 (1978)), slaughtered cattle brains in ice-cold isotonic phosphate-buffered saline are mechanically freed from blood vessels or larger blood clots and frozen in liquid nitrogen. After thawing, homogenization of the brains is carried out at pH 4.5 in a 0.15 M (NH ₄ ) ₂ SO ₄ solution. After 2 hours of stirring and subsequent centrifugation (23000g, 45 min.), The recovered supernatant is adjusted to pH 6.5 to 7.0 with 1 N NaOH and subjected to (NH ₄ ) ₂ SO ₄ precipitation (1.66 M). After centrifugation, the supernatant now obtained is subjected to a further (NH ₄ ) ₂ SO ₄ precipitation (3.56M). The sediment resulting from subsequent centrifugation is taken up in distilled water and subjected to desalting dialysis against distilled water. After centrifugation of the resulting sediment, the clear centrifugation supernatant is lyophilized. AII operations are performed at 4 ⁰ C.

The proteinaceous preparation obtained in this way possesses, in addition to "fibroblast growth factor" activity, other unspecified components which promote the cell growth-promoting interaction of "fibroblast growth factor" and serum components in a manner which results in a hitherto unknown long-lasting effectiveness This growth factor ("Fibroblast Growth Factor") results in the case - cultivation of cells of mesodermal origin, in particular human amniotic fluid cells - makes it possible to reduce the otherwise required frequent medium renewal during cell culture.

Example 2: Preparation of the medium admixture or of the medium for cultivating human amniotic fluid cells

The brain dry preparation obtained according to Example 1 is dissolved in a little distilled water. After determining the protein content of this solution (protein determination method LOWRY) this is adjusted by further addition of distilled water so that an easily manageable dosage of the provided for lyophilization in ampoules of protein amount is possible. It is expediently so dimensioned that with a single admixture of the contents of an ampoule to a previously prepared for immediate use standard amount of supplementation for a cell culture medium (eg, 11, 10% (v / v) containing fetal calf serum) the optimal concentration of brain preparation ΠΟΟμ / ml) in the ready-to-use medium.

In remelted under nitrogen vials on storage at -20 ⁰ C with respect to the biological activity of cell proliferation in the brain dry preparation is maintained for at least 2 years.

Example 3: Demonstration of the influence of the brain preparation on the proliferation of human amniotic fluid cells

In a test arrangement representative of primary cultures, human amniotic fluid cells of the second to ninth subcultures are used for propagation at a seeding density of 500 cells per petri dish (diameter: 6 cm) using the usual techniques of cell cultivation. The medium used contains as a basal medium commercial EAGLE (MEM) cell culture medium with 0.5% (w / v) lactalbumin hydrolyzate, to which different amounts of fetal calf serum and the described brain solid preparation were added. With a single change of medium on the 7th day after the beginning of the test, the evaluation of the experimental batches took place on the 14th day of the experiment (Table 1 and 2). Table 1: The effectiveness of different amounts of brain dry preparation in a basal medium + 10% fetal calf serum cell culture medium on the cultivation of human amniotic fluid cells ZusatzanHirn- On the 14th experimental day determined

dry preparation Cell count per Petri dish Relative plating

Efficiency (%)

0 33800 ± 29100 14.8 ± 18.7

50 55 600 ± 50 000 81.0 ± 14.4

100 180000 ± 103000 100.0 + 0

200 83800+ 44600 59.2 + 22.2

500 112 500+ 58600 27.0 ± 19.6

1000 - 0.75 ± 1.5

Table 2: Influence of different amounts of fetal calf serum in the presence of brain dry preparation (optimal concentration) on the plating efficiency in the cultivation of human amniotic fluid cells

Serum Concentration in Concentration of Plating Efficiency

Medium /% (v / v) / brain-dried preparation (%)

(Μq / rτ \\)

20 0 5.1 + 0.9

0 100 0

5 100 6.8 + 1.1

10 100 9.2 + 0.7

15 100 10.4 ± 0.6

20 100 11.0 + 0.9

As shown in Tables 1 and 2, optimum conditions exist in terms of the relative plating efficiency as well as the increase in cell proliferation when 10% fetal calf serum and 100 / g / ml of brain extract are added to the basal medium. Under these conditions, for example, by the brain preparation, an approximately 5.4-fold increase in cell yield can be achieved, so this method meets the requirements of prenatal diagnosis - with simultaneous advantages in terms of medium exchange frequency - justice. Without adding serum to the medium, the brain preparation is ineffective, however, when used, the amount of fetal calf serum in the medium, which is 20% by conventional methods, can be reduced by 50% (Table 2).

Based on comparable references (GOS-PODAROWICZ, MORAN and OWASHI, J. Clin Endocrinol, Metab., 44, 651 (1977)), the proliferation rate of human amniotic fluid cells achievable with the medium described herein is also due to the action of a medium consisting of modified EAGLE medium DULBECCO (DME) and 20% (v / v) fetal calf serum - superior.

In the amniotic fluid there are essentially three morphologically distinguishable cell types (fibroblastoid and epitheloid cells as well as "amniotic fluid" cells.) As shown in Table 3, the medium described here has a positive effect on the growth of all three cell types.

Table 3: The effect of brain preparations to the cell culture medium (basal medium + 10% fetal calf serum) on the plating efficiency of various morphological cell types in the cultivation of human amniotic fluid cells Cell type Plating Efficiency (%)

in the presence of in the absence of

Hirntrockenpräparat Hirntrockenpräparat

(100 ug / ml)

Fibroblastoid 25.5 ± 1.0 16.3 + 1.0

Amniotic fluid type 8,4 ± 0,5 3,2 + 0,6

Epithelioid type 4.4 ± 0.6 0

When using the medium described here, ie under the influence of the brain preparation, important parameters of the amniotic fluid lines, such. B. the activities of the enzymes a-galactosidase, ß- galactosidase, / 3-glucuronidase and arylsulfatase A is not affected.

Claims (1)

Cell culture medium for prenatal diagnosis, characterized in that it consists of EAGLE (MEM) cell culture medium and 0.5% (w / v) lactalbumin hydrolyzate to which 10% (v / v) fetal calf serum has been added and further 100μg / ml of a proteinaceous Bovine brain dry preparations obtained under sterile working conditions a) mechanical removal of larger blood vessels and blood clots from slaughter-warm cattle brains in ice-cold isotonic phosphate-buffered saline, germination of the brain tissue by placing in liquid nitrogen, thawing and homogenization in a 0.15M (NH ₄ ) SO ₄ solution at pH 4.5, stirring for two hours, and subsequent centrifugation, b) a (NH ₄ ) ₂ S0 ₄ precipitation of the centrifugation supernatant at pH 6.5 to 7.0 and centrifugation again, c) a further (NH ₄ ) ₂ S0 ₄ precipitation of the supernatant and further centrifugation, d) recording of the sediment of the latter centrifugation in distilled water, a desalting dialysis of this solution against distilled water, centrifugation of the resulting sediment and lyophilization of the clear Zentrifugationsüberstandes and which freshly mixed before use under sterile working conditions.