DD225440A1 - CELL MEDIUM BREED - Google Patents

Abstract

The invention relates to a cell culture medium, in particular for use in prenatal diagnosis. According to the invention, this cell culture medium contains, in addition to usual constituents, a preparation obtained from the brain of freshly slaughtered cattle, which in addition to components which are not further defined contains "fibroblast growth factor" activity. Due to a synergism between "fibroblast growth factor" and the undefined accompanying components, preferably consisting of proteins, used in an optimal concentration, this brain preparation, together with usual medium components, has a sustained promoting effect on the proliferation of cells, in particular small quantities of human amniotic fluid cells in vitro , By means of the invention, it is possible to increase small amounts of human amniotic fluid cells, as are customarily produced in amniotic fluid samples, in a time period which is reasonable for prenatal diagnosis, or to the required extent without major technical outlay.

Description

Field of application of the invention

The invention relates to a cell culture medium containing in addition to conventional components obtained from a brain freshly slaughtered cattle preparation, whereby the proliferation of cells of mesodermal origin, especially human amniotic fluid cells - especially at low Zeileinsaat amount - 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

Known is the possibility of very small amounts of animal and human cells, such as those incurred in amniotic fluid samples in the context of pranatal diagnostics, by conventional Zeilzuchtmedien which is added as a growth-promoting component up to 20 ³ O fetal Käiberserum in vitro to increase , In certain cases, oei which the time factor for the achievement of a suitable or required for the re-use of the cells amount of cells - such. For examinations ^; In the prenatal diagnostic field, other adjuncts of cell growth factors to the medium have been reported, in particular additions of "fibroblast growth factor" in substantially purified form (1 μg / ml, PORRECO, 3RADSHAW, SARKAR and JONES, Obstetrics and Gynecology 55, 55 (1980)) or in pure form (100 μg / ml) (GOSPODAROVICZ, MORAN and OWASHI, J. Cün. Endocrinol.

Metab. 44, 551 1977}) have proven to be generally successful, but here - as in the latter case - the cell seeds intended for propagation are relatively high (about 3,000 cells per Petri dish / scm diameter /), and furthermore in the cases mentioned cell cultivation with the technical disadvantage associated with daily or very frequently 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 that it usually possesses 'GOSPODAROVICZ and MORAN, Annual Rev. Biochem., 451, 531 (1976)) are partially available from the serum additions to the cell culture medium. Thus, the multiple medium change required in the abovementioned cases, addition of consumed active substance reserves, is also explained, with its frequency an infectious biological risk increases m the case of the cultivation of human amniotic fluid cells in a special way, which is that a possibly connected to the medium renewal Mykouiasmeninfektion the amniotic fluid lines to the same chromosomal changes - and ^2u, - from the technical disadvantage work Diagnostic Mistakes - Can Lead! SCHNEIDER, STAiMBRIDGE, EPSTEIN, GOLBUS. ABBO-HALSBASCH and RODGERS, Science 184, 477 (1974)).

Also with the aim of reducing the risk of mycoplasma contamination - here, however, preferably in the. causative relationship with higher serum concentrations (15 to 30%) ^: seen in the special case of the propagation of human amniotic fluid lines in vitro successful examinations for a substantial substitution of fetal calf serum .m medium by a "fibroblast growih factor" in conjunction with 9 other biofactors, predominantly hormones) have become known, but here, too, serum supplementation of fetal calf serum;, -on - ^: -, is to be reduced to medium. " ¹ ANG JONES and MASUI Proc. Nat. Acad USA 79, 4795 11982. 'In this case as well, more frequent renewal of the medium during the cultivation of Zail can not be dispensed with. NORTHOFF (DE-OS 3,138,597) claims to accelerate the growth of amniotic fluid lines or of human Written in which the stability of pure "fibroblast growth factor" as well as its solubility is judged critically and a better effect of an admixture of a

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lymphokine-rich supernatant of concavalin A stimulated lymphocytes 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 cell culture media.

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 is used by mixing this brain preparation (dry preparation) in an optimal concentration (ΙΟΟμ-g / ml) to a conventional cell culture medium (eg., EAGLE cell culture medium / MEM /) containing 1O ⁰ O (v / v) fetal calf serum , a novel composite cell culture medium is formed, with the one of the requirements of prenatal cytogenetic and biochemical diagnostics in an improved way fair increase in human amniotic fluid cells is made possible, especially the otherwise required frequency of adverse medium renewal can be undershot.

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 0.15M (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. All operations are carried out at 4 ° 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"), which makes it possible in the given case-cultivation of cells of mesodermal origin, in particular human amniotic fluid cells - a reduction of 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 admixing of the contents of an ampoule with a standard amount previously prepared for immediate use in a cell culture medium intended for supplementation (eg containing 11, 10% (v / v) fetal calf serum) the optimal concentration Brain preparation (ΙΟΟμ, / ml) is ensured in the ready medium.

In ampoules sealed under nitrogen, when stored at -20 ° C., the biological activity for cell proliferation in the brain-dried preparation is maintained for at least 2 years.

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

In an experimental setup representative of primary cultures, human amniotic fluid cells of the second to ninth subcultures are seeded at a seeding density of 500 cells per petri dish (diameter: 6 cm) for multiplication using the usual techniques of cell culture. 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 efficacy of varying amounts of brain dry preparation in a cell culture medium consisting of basal medium + 10% fetal calf serum on the cultivation of human amniotic fluid cells

Additional to Brain On the 14th day of testing

dry preparation Cell count per Petri dish Relative plating

(/ xg / ml) Efficiency (%)

0 33800 ± 29100 14.8 ± 18.7

50 55 600 ± 50 000 81.0 ± 14.4

100 180000 ± 103000 100.0 ± 0

200 83 800+ 44 600 59.2 ± 22.2

500 112 500 ± 58 600 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 the concentration of plating efficiency

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

(/ Ig / ml)

20 0 5,1x0,9

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, when the cells are attached to the basal medium with 10% fetal calf serum and 100 μg / ml of the brain dry preparation, the conditions of relative plating efficiency and increase of cell proliferation are optimal. 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).

On the basis of comparable references (GOS-PODAROWICZ, MORAN and OWASHI, J. Clin. Endocrinol.

Metab. 44,651 (1977)), the reproduction rate of human amniotic fluid cells achievable with the medium described here is also superior to the action of a medium consisting of modified EAGLE medium according to DULBECCO (DME) and 20% (v / v) fetal calf serum.

In the amniotic fluid there are essentially three morphologically distinguishable cell types (fibrobiastoid 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 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

(100Aig / ml)

Fibroblastoid 25.5 ± 1.0 16.3 + 1.0

Amniotic fluid cell 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, are important parameters of the amniotic fluid cells, such as, for example, for biochemical prenatal diagnosis. B. the activities of the enzymes α-galactosidase, ß- galactosidase, β-glucuronidase and arylsulfatase A are not affected.

Claims (3)

-1- 261 242 1 Invention claims: Cell culture medium, 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 ΙΟΟμ / ml of a proteinaceous dry preparation from bovine brain, 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 0.15 M (NH ₄ ) ₂ SO ₄ solution at pH 4.5, two hours of stirring and subsequent centrifugation, b) a (NH ₄ J ₂ SO ₄ -FiIlUrIg centrifugation supernatant at pH 6.5 to 7.0 and re-centrifugation, c) a further (NH ₄ ) ₂ SO ₄ precipitation of the supernatant and further centrifugation, d) recording 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 to be mixed freshly before use under sterile working conditions. 2. cell culture medium according to item 1, characterized in that it brings low amounts of cells of mesodermal origin when it falls below the usually required frequency of medium renewal for multiplication when using conventional zeilzüchterischer working methods. 3. cell culture medium according to item 1 and 2 for use in prenatal diagnosis, characterized in that it contains small amounts of human amniotic fluid cells, as obtained in prenatal diagnostics in amniotic fluid samples, in a prenatal for the period of 14 to 28 days up to a sufficient number of cells for routine genetic examinations - approximately 10 ⁴ to 10 ⁶ cells - brings about an increase in all the amniotic fluid in the amniotic fluid, without adversely affecting important cell biochemical enzyme parameters and undercutting the usually required frequency of medium renewal.