DE3713197A1 - Method for the long-term culture of intervertebral discs and cell cultures which can be isolated therefrom - Google Patents
Method for the long-term culture of intervertebral discs and cell cultures which can be isolated therefromInfo
- Publication number
- DE3713197A1 DE3713197A1 DE19873713197 DE3713197A DE3713197A1 DE 3713197 A1 DE3713197 A1 DE 3713197A1 DE 19873713197 DE19873713197 DE 19873713197 DE 3713197 A DE3713197 A DE 3713197A DE 3713197 A1 DE3713197 A1 DE 3713197A1
- Authority
- DE
- Germany
- Prior art keywords
- intervertebral discs
- long
- cell cultures
- intervertebral
- term culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 238000000034 method Methods 0.000 title claims abstract description 8
- 238000004113 cell culture Methods 0.000 title claims abstract description 7
- 230000007774 longterm Effects 0.000 title claims description 7
- 108060005980 Collagenase Proteins 0.000 claims description 11
- 102000029816 Collagenase Human genes 0.000 claims description 11
- 235000015097 nutrients Nutrition 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 238000009877 rendering Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- 229960002424 collagenase Drugs 0.000 description 5
- 239000012980 RPMI-1640 medium Substances 0.000 description 4
- 239000012983 Dulbecco’s minimal essential medium Substances 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 239000001569 carbon dioxide Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000193159 Hathewaya histolytica Species 0.000 description 1
- 108010013295 Microbial collagenase Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 235000002639 sodium chloride Nutrition 0.000 description 1
- 238000013337 sub-cultivation Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/066—Tenocytes; Tendons, Ligaments
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0655—Chondrocytes; Cartilage
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Rheumatology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Rehabilitation Therapy (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
Es sind bereits Kultursysteme für Gewebe verschiedener Herkunft bekannt (R. Ian Freshney: Culture of animal cells, A Manual of Basic Technique, Alan R. Liss, Inc., New York 1983, ISBN 0-8451-0223-0, Seiten 104ff). In solchen Kultursystemen hält sich das Gewebe jedoch nur dann über einen längeren Zeitraum, wenn es in sehr kleine Teile (größter Durchmesser unter 1 bis 2 mm) zerschnitten worden ist (loc.cit. Seiten 5 und 225). Die bislang bekannten Methoden versagen bei Geweben mit den Ausmaßen einer Bandscheibe (z.B. des Hundes) und führen nach kurzer Zeit zum Absterben der innenliegenden Zellen. Die Langzeitkultivierung von Bandscheibengewebe wurde bisher nicht bekannt.Culture systems for tissues of various origins are already known (R. Ian Freshney: Culture of animal cells, A Manual of Basic Technique, Alan R. Liss, Inc., New York 1983, ISBN 0-8451-0223-0, pages 104ff). In such culture systems, however, the tissue only survives over one longer period if it is in very small parts (largest diameter under 1 to 2 mm) has been cut (loc.cit. Pages 5 and 225). The Previously known methods fail in the size of a tissue Intervertebral disc (e.g. of the dog) and lead to death after a short time of the cells inside. The long-term cultivation of intervertebral disc tissue has so far not been known.
Es wurde nun ein Weg gefunden, um Bandscheiben unter Erhalt ihrer biologischen Funktionen über lange Zeit zu kultivieren und daraus vermehrungsfähige Zellkulturen herzustellen.A way has now been found to preserve their intervertebral discs to cultivate biological functions over a long period and from them to produce viable cell cultures.
Gegenstand der Erfindung ist ein Verfahren zur Langzeitkultur von Band scheiben, welches darin besteht, daß man die Bandscheiben einem Voll nährmedium aufbewahrt.The invention relates to a method for the long-term culture of ribbon discs, which consists in that the intervertebral discs a full stored nutrient medium.
Gegenstand der Erfindung ist weiter die Herstellung von Bandscheibenzell kulturen, welches dadurch gekennzeichnet ist, daß man in einem Vollnähr medium kultivierte Bandscheiben nach frühestens 5 Tagen Kultivierungszeit mit Kollagenase in die einzelnen Zellen zerlegt und diese in an sich bekannter Weise vermehrt.The invention further relates to the production of intervertebral disc cells cultures, which is characterized by the fact that one in a whole medium cultured intervertebral discs after a cultivation period of at least 5 days with collagenase broken down into the individual cells and these into themselves known manner increased.
Das für die Langzeitkultivierung von Bandscheiben verwendete Vollnähr medium muß alle essenziellen Aminosäuren, Spurenelemente und Salze sowie Glucose enthalten. Es besitzt vorzugsweise einen pH-Wert von etwas unter 7,4. Solche Vollnährmedien sind im Handel beispielsweise unter den Bezeichnungen Ham F 12, Dulbecco′s Minimal Essential Medium und RPMI 1640 erhältlich. Diesen Medien muß noch 2 bis 20, vorzugsweise 5 bis 10% eines Serums zugesetzt werden. Besonders eignet sich als Zusatz eine Mischung aus etwa gleichen Teilen foetalem Kälberserum und Pferdeserum.The complete nutrient used for the long-term cultivation of intervertebral discs medium must contain all essential amino acids, trace elements and salts as well Contain glucose. It preferably has a pH slightly below 7.4. Such complete nutrient media are commercially available, for example, among the Designations Ham F 12, Dulbecco’s Minimal Essential Medium and RPMI 1640 available. These media must still have 2 to 20, preferably 5 to 10% of one Serums can be added. A mixture is particularly suitable as an additive from approximately equal parts of fetal calf serum and horse serum.
Die Langzeitkultivierung von Bandscheiben wird noch wesentlich verbessert, wenn man diese im Nährmedium unter einer Atmosphäre aufbewahrt, deren Sauerstoffgehalt vermindert und deren Kohlendioxidgehalt erhöht ist. Besonders günstige Resultate werden erzielt, wenn der Sauerstoffgehalt auf bis zu 21%, vorzugsweise auf 5 bis 10%, reduziert und der Kohlendioxid gehalt auf 5 bis 20%, vorzugsweise 10 bis 15% erhöht wird. The long-term cultivation of intervertebral discs is significantly improved, if you keep them in the culture medium under an atmosphere whose Oxygen content is reduced and its carbon dioxide content is increased. Particularly favorable results are achieved when the oxygen content is on up to 21%, preferably to 5 to 10%, and reduced the carbon dioxide content is increased to 5 to 20%, preferably 10 to 15%.
Die so behandelten Bandscheiben lassen sich durch Spaltung mit Kollagenasen in Zellen zerlegen. Diese Spaltung führt jedoch frühestens nach einer Kultivierungszeit der Bandscheiben im Vollmedium nach etwa 5 Tagen zu brauchbaren Zellkulturen. Am besten gelingt die Spaltung in Einzelzellen ab dem 7. Tag.The intervertebral discs treated in this way can be split by Break down collagenases into cells. However, this split leads at the earliest after a culture period of the intervertebral discs in the full medium after about 5 days to usable cell cultures. The split is best Single cells from the 7th day.
Anstelle der Kollagenasen lassen sich auch andere Proteasen verwenden, jedoch werden mit Kollagenasen die besten Ergebnisse erzielt. Als Kollagenasen können die im Handel erhältlichen Rohkollagenasen aus Clostridium histolyticum verwendet werden.Instead of the collagenases, other proteases can also be used, however, the best results are achieved with collagenases. As Collagenases can be obtained from the commercially available raw collagenases Clostridium histolyticum can be used.
Vor der Behandlung mit Kollagenase wird die Bandscheibe zweckmäßig mechanisch zerkleinert.The intervertebral disc is appropriate before treatment with collagenase mechanically crushed.
Die Behandlung mit Kollagenase wird vorzugsweise in dem Medium durchgeführt, in dem die Bandscheibe haltbar gemacht wurde. Die Rohkollagenase wird in der Regel in einer Menge von 1 mg/ml für 200 mg Bandscheibengewebe eingesetzt. Die Spaltung des Bandscheibengewebes gelingt am besten bei 35 bis 39, vorzugsweise 37°C und bei einem pH-Wert von 5 bis 7,4. Die Spaltung wird zweckmäßig unter Schütteln durchgeführt und wird in der Regel nach 10 bis 20 h beendet.Treatment with collagenase is preferably in the medium performed in which the intervertebral disc was preserved. The Raw collagenase is usually used in an amount of 1 mg / ml for 200 mg Intervertebral disc tissue inserted. The splitting of the intervertebral disc tissue works best at 35 to 39, preferably 37 ° C and at a pH from 5 to 7.4. The cleavage is expediently carried out with shaking and is usually ended after 10 to 20 hours.
Die Erfindung ermöglicht es, Bandscheibenzellen in großer Menge für wissenschaftliche Zwecke zur Verfügung zu stellen. Weiter wird es durch die Erfindung möglich, Faktoren zu suchen, die zur Behandlung von Bandscheibenschäden geeignet sind.The invention enables disc cells to be used in large quantities to provide scientific purposes. It continues through the invention makes it possible to search for factors that are used to treat Intervertebral disc damage is suitable.
Das erfindungsgemäße Verfahren ermöglicht es, kontinuierlich die Wechsel wirkungen zwischen Bandscheibe und Pharmaka mit nichtinvasiven Methoden zu untersuchen, z.B. nach Enzymbehandlung enzymatische Reaktionsprodukte aus dem Kulturmedium zu bestimmen. Durch dieses in-vitro-Modell werden systemische Einflüsse - etwa durch die Leber - ausgeschaltet, wobei jedoch die zelluläre Organisation im Gewebe erhalten bleibt. Außerdem läßt sich der Einfluß lytisch wirkender Enzyme auf die intakte interzellulare Matrix untersuchen. Eine solche Untersuchung ist mit biochemischen Methoden bislang nicht möglich gewesen.The method according to the invention makes it possible to continuously investigate the interactions between the intervertebral disc and pharmaceuticals using non-invasive methods, for example to determine enzymatic reaction products from the culture medium after enzyme treatment. This in vitro model eliminates systemic influences - such as those caused by the liver - while maintaining the cellular organization in the tissue. In addition, the influence of lytic enzymes on the intact intercellular matrix can be examined. Such an investigation has so far not been possible with biochemical methods.
Lumbale Bandscheiben von Kaninchen (50 bis 300 mg) wurden in einer Petrischale in 5 ml Vollmedium gebracht. Das Vollmedium bestand aus 1 Teil Dulbecco′s Minimal Essential Medium, 1 Teil Ham′s F 12 und 2 Teilen RPMI 1640, welche je 5% fötales Kälberserum und Pferdeserum enthielten. Zur Vermeidung eines bakteriellen Befalls wurde Penicillin zugesetzt.Lumbar discs from rabbits (50 to 300 mg) were placed in 5 ml of complete medium in a petri dish. The full medium consisted of 1 part Dulbecco's Minimal Essential Medium, 1 part Ham's F 12 and 2 parts RPMI 1640, each containing 5% fetal calf serum and horse serum. Penicillin was added to prevent bacterial infection.
Die Kulturen wurden bei 36,5°C gehalten. Die umgebende Luft war mit Kohlendioxid (ad 10%) und mit Stickstoff so angereichert, daß der Sauerstoffgehalt auf 10% abgesenkt war. Die Luftfeuchtigkeit betrug über 90%. Die Bandscheiben lassen sich so mindestens 3 Monate halten.The cultures were kept at 36.5 ° C. The surrounding air was with Carbon dioxide (ad 10%) and enriched with nitrogen so that the Oxygen content was reduced to 10%. The air humidity was over 90%. The intervertebral discs can be kept for at least 3 months.
Das gleiche Ergebnis erhält man, wenn man anstelle des Gemisches aus Dulbecco′s Minimal Essential Medium, Ham′s F 12 und RPMI 1640 das Medium RPMI 1640 alleine verwendet.The same result is obtained if, instead of the mixture of Dulbecco's Minimal Essential Medium, Ham's F 12 and RPMI 1640, the medium RPMI 1640 is used alone.
Nach 1wöchiger und 3monatiger Kultur wurden Bandscheiben aus der Kultur entnommen und mit einem Skalpell in kleine Stücke zerschnitten. Aus Roh kollagenase von Clostridium histolyticum wurde mit oben genanntem Voll medium eine Lösung (1 mg/ml) angesetzt. Bandscheiben unter 200 mg wurden in 1 ml der Kollagenaselösung suspendiert, bei größeren Bandscheiben wurden 1,5 ml eingesetzt. Die Suspension wurde in einem Wasserbad bei 36,5°C 14 h geschüttelt. Freigesetzte Zellen und Zellagglomerate wurden mit einem Sieb von restlichen Gewebsstücken getrennt. Die abgetrennten Zellen wurden in Zellkultur-Petrischalen unter den für die Bandscheiben beschriebenen Bedingungen gehalten. Die Subkultivierung gelingt mit den üblichen Maßnahmen.After 1 week and 3 months of culture, discs were removed from the culture removed and cut into small pieces with a scalpel. From raw Clostridium histolyticum collagenase was obtained with the abovementioned Voll medium prepared a solution (1 mg / ml). Intervertebral discs were below 200 mg suspended in 1 ml of the collagenase solution, for larger intervertebral discs 1.5 ml were used. The suspension was added in a water bath Shaken at 36.5 ° C for 14 h. Released cells and cell agglomerates were separated from remaining pieces of tissue with a sieve. The severed Cells were placed in cell culture petri dishes under those for the intervertebral discs described conditions kept. Subcultivation works with the usual measures.
Claims (2)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19873713197 DE3713197A1 (en) | 1987-04-18 | 1987-04-18 | Method for the long-term culture of intervertebral discs and cell cultures which can be isolated therefrom |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19873713197 DE3713197A1 (en) | 1987-04-18 | 1987-04-18 | Method for the long-term culture of intervertebral discs and cell cultures which can be isolated therefrom |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| DE3713197A1 true DE3713197A1 (en) | 1988-11-03 |
Family
ID=6325904
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DE19873713197 Withdrawn DE3713197A1 (en) | 1987-04-18 | 1987-04-18 | Method for the long-term culture of intervertebral discs and cell cultures which can be isolated therefrom |
Country Status (1)
| Country | Link |
|---|---|
| DE (1) | DE3713197A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5422261A (en) * | 1993-04-16 | 1995-06-06 | Baxter International Inc. | Composition containing collagenase and chymopapain for hydrolyzing connective tissue to isolate cells |
| US5670358A (en) * | 1995-10-19 | 1997-09-23 | Baxter International Inc. | Method for inhibiting chymopapain and papain enzyme activity with polysaccharides of animal origin |
-
1987
- 1987-04-18 DE DE19873713197 patent/DE3713197A1/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5422261A (en) * | 1993-04-16 | 1995-06-06 | Baxter International Inc. | Composition containing collagenase and chymopapain for hydrolyzing connective tissue to isolate cells |
| US5424208A (en) * | 1993-04-16 | 1995-06-13 | Baxter International Inc. | Method for isolating cells from tissue with a composition containing collagenase and chymopapin |
| US5670358A (en) * | 1995-10-19 | 1997-09-23 | Baxter International Inc. | Method for inhibiting chymopapain and papain enzyme activity with polysaccharides of animal origin |
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| Date | Code | Title | Description |
|---|---|---|---|
| 8141 | Disposal/no request for examination |