Process for the preparation of defibrinated and lyophilized placental cells

• US 4,193,992 A
• Filed: 12/21/1977
• Issued: 03/18/1980
• Est. Priority Date: 08/16/1977
• Status: Expired due to Term

• Current Assignee: Laboratories Cellorgan SA
• Original Assignee: Laboratories Cellorgan SA
• Inventors: Fontaine, Gerard
• Primary Examiner(s): Robinson, Allen J.
• Application Number:
  US05/862,911
• Time in Patent Office: 818 Days
• Field of Search: 424/105, 195/1.8, 195/2, 195/4
• US Class Current: 424/583
• CPC Class Codes: A61K 35/50 Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
• CPC Class Codes: A61K 8/982 Reproductive organs; Embryos, Eggs
• CPC Class Codes: A61Q 19/00 Preparations for care of the skin

Abstract

Defibrinated and lyophilized placental cells are prepared by taking the foetomaternal placenta of a ewe killed at the end of the fourth month of gestation, breaking the placenta into fragments, immediately submerging the fragments in Ringer'"'"'s solution to wash away traces of blood, finely dividing the fragments, placing them in 5% aqueous trypsinated solution, permitting tryptic digestion with stirring, separating the placental cells by filtering, washing the cells in refrigerated Hanks solution, centrifuging in a low temperature centrifuge, suspending the base obtained in the centrifuging step in a minimum of survival liquid and lyophilizing the suspension until constant weight is obtained. The cells obtained are used in the external treatment of ulcers and first and second degree burns.

Claims

1. A process for the preparation of defibrinated and lyophilized placental cells comprising breaking into fragments the foetomaternal placenta of a ewe killed at about the end of the fourth month of gestation, immediately submerging the fragments in Ringer'"'"'s solution in order to wash away traces of blood, finely dividing the fragments and placing them in a 1 to 10% aqueous trypsinated solution, effecting tryptic digestion in said solution with the aid of stirring, separating the placental cells by filtering, washing the cells in refrigerated Hanks medium and separating the cells by centrifuging at a low temperature, suspending the cellular residue obtained from the centrifuging process in survival liquid and lyophilizing the suspension until a constant weight is obtained.

2. Defibrinated lyophilized placental cells prepared according to the process of claim 1.

5. A process according to claim 1 wherein the amount of survival liquid is the minimum required to permit lyophilization of the suspended base.

6. A process according to claim 1 wherein the fragments are placed in a 5 weight % aqueous trypsinated solution.

7. A process according to claim 1 wherein the washing and centrifuging are carried out at 10°

to 40°

C.

8. A process according to claim 7 wherein there is employed 5% aqueous trypsinated solution, the tryptic digestion is carried out at 38°

C. for 2 hours, the cells are washed 3 times in the refrigerated Hanks medium, centrifuging is carried out at 1,000 rpm and at 38°

C.

9. A process according to claim 8 including the further step of portioning out the lyophilized product into ampules sealed under nitrogen or vacuum.

3. Defibrinated, lyophilized placental cells soluble in water to the extent of 0.14 grams in 5 ml at 20°

C., a 1% solution of said cells having a pH of about 7.3-7.7, a 2% aqueous solution of the cells having a resistivity of about 229.8±

33.7 ohms cm² /cm, said cells having intense absorption within the range of 200 to 225 nm and also marked absorption in the range between 225 and 280 nm, an ash content of 9.37±

0.48%, a total nitrogen content of 10.17±

0.38%, a non-protein nitrogen content of 3.75±

0.28%, a sodium content of 32.23±

6.19 mg/g, a potassium content of 3.32±

0.50 mg/g, a calcium content of 10.65±

mg/g, a magnesium content of 1±

0.17 mg/g, a zinc content of 0.12±

0.01 mg/g, a chlorine content of 30.80±

3.41 mg/g and a phosphorus content of 4.05±

0.37 mg/g.

4. Defibrinated, lyophilized placental cells according to claim 3 characterized by the ability to be obtained by breaking into fragments the foetomaternal placenta of a ewe killed at about the end of the fourth month of gestation, immediately submerging the fragments in Ringer'"'"'s solution in order to wash away traces of blood, finely dividing the fragments and placing them in a 5% aqueous trypsinated solution, effecting tryptic digestion in said solution with the aid of stirring, separating the placental cells by filtering, washing the cells in refrigerated Hanks medium and separating the cells by centrifuging at a low temperature, suspending the base obtained from the centrifuging process in survival liquid and lyophilizing the suspension until a constant weight is obtaines.