A PROCESS OF PREPARING BUCCAL EPITHELIAL CELL SUSPENSION AND ITS USE

Abstract

A process of preparing buccal epithelial cell suspension and cystoscopically implanting the cell suspension in the defect site of the adult human urethra.

Claims

1. A process of preparing autologous buccal epithelial cell suspension comprising (a) harvesting buccal mucosal tissue from subject; (b) treating the tissue from (a) with chemical dissociation agent; (c) washing the tissue sample from (b) with nutrient medium; (d) separating the epidermis from dermis of the washed tissue; (e) mincing, filtering to obtain uniform epithelial cell suspension from epidermis; (f) optionally seeding to enable cell multiplication; (g) harvesting the cellular monolayer with enzyme(s); (h) centrifuging, discarding the supernatant; (i) mixing with nutrient medium; (j) analyzing the cell suspension; (k) filling 0.4 ml of the cell suspension in V shaped 1 ml vials; and (l) optionally transporting to the same subject as in (a).

2. A process of preparing autologous buccal epithelial cell suspension as claimed in claim 1 wherein the subject is an adult human subject.

3. A process of preparing autologous buccal epithelial cell suspension as claimed in claim 1 further comprising optionally mixing with gel while cystoscopically implanting the cell suspension into the defect site of the urethra of the subject; wherein the gel is selected from biocompatible delivery system such as combination of fibrinogen and/or thrombin and/or thermo-reversible gelation polymer (TGP) gel and/or collagen and/or chitosan and the like.

4. A process of preparing autologous buccal epithelial cell suspension as claimed in claim 1 wherein the chemical dissociation agent is selected from trypsin, dispase, collagenase, trypsin-EDTA, pronase, hyaluronidase, elastase, papain and pancreatin.

5. A process of preparing autologous buccal epithelial cell suspension as claimed in claim 1 wherein the nutrient medium is selected from DMEM, EMEM, F12, IMDM and the like.

6. A process of preparing autologous buccal epithelial cell suspension as claimed in claim 1 wherein the seeding is done in T-25 and/or T-75 and/or T-150 flask and the like.

7. A process of preparing autologous buccal epithelial cell suspension as claimed in claim 1 wherein the enzyme for harvesting is selected from trypsin-EDTA, collagenase and the like.

8. A process of preparing autologous buccal epithelial cell suspension as claimed in claim 1 wherein the analysis performed are Appearance, Sterility, Mycoplasma, Endotoxin, Cell Counting, Cell Viability, Cell Purity Test, Cell Characterization and Karyotyping Analysis.

9. A process of preparing autologous buccal epithelial cell suspension as claimed in claim 1 wherein the harvested buccal mucosal tissue is from the inner check of adult human and is about 11.5 cm.sup.2.

10. A process of preparing autologous buccal epithelial cell suspension as claimed in claim 1 wherein the transportation is at 2 to 8 degree centigrade.

11. A method of cystoscopically implanting the autologous buccal epithelial cell suspension into the defect site of the adult human urethra comprising (a) harvesting human buccal mucosal tissue from the inner cheek of the adult human; (b) subjecting the tissue from (a) to chemical dissociation agent; (c) washing the tissue sample from (b) with nutrient medium; (d) separating the epidermis from dermis of the washed tissue; (e) mincing, filtering to obtain uniform epithelial cell suspension from epidermis; (f) optionally seeding to enable cell multiplication; (g) harvesting the cellular monolayer with enzyme(s); (h) centrifuging, discarding the supernatant; (i) mixing with nutrient medium; (j) analyzing the cell suspension; (k) optionally filling and transporting the cell suspension in V shaped 1 ml vials; and (l) optionally mixing with gel while cystoscopically implanting the autologous buccal epithelial cell suspension into the defect site of the adult human urethra.

12. A method of cystoscopically implanting the autologous buccal epithelial cell suspension into the defect site of the adult human urethra as claimed in claim 11 wherein the chemical dissociation agent is selected from trypsin, dispase, collagenase, trypsin-EDTA, pronase, hyaluronidase, elastase, papain and pancreatin.

13. A method of cystoscopically implanting the autologous buccal epithelial cell suspension into the defect site of the adult human urethra as claimed in claim 11 wherein the nutrient medium is selected from DMEM, EMEM, F12, IMDM and the like.

14. A method of cystoscopically implanting the autologous buccal epithelial cell suspension into the defect site of the adult human urethra as claimed in claim 11 wherein the seeding is done in T-25 and/or T-75 and/or T-150 flask and the like.

15. A method of cystoscopically implanting the autologous buccal epithelial cell suspension into the defect site of the adult human urethra as claimed in claim 11 wherein the enzyme is selected from trypsin-EDTA, collagenase and the like.

16. A method of cystoscopically implanting the autologous buccal epithelial cell suspension into the defect site of the adult human urethra as claimed in claim 11 wherein the analysis performed are Appearance, Sterility, Mycoplasma, Endotoxin, Cell Counting, Cell Viability, Cell Purity Test, Cell Characterization and Karyotyping Analysis.

17. A method of cystoscopically implanting the autologous buccal epithelial cell suspension into the defect site of the adult human urethra as claimed in claim 11 wherein the harvested human buccal mucosal tissue from the inner cheek of the human is about 11.5 cm.sup.2.

18. A method of cystoscopically implanting the autologous buccal epithelial cell suspension into the defect site of the adult human urethra as claimed in claim 11 wherein the transportation is at 2 to 8 degree centigrade.

19. A method of cystoscopically implanting the autologous buccal epithelial cell suspension into the defect site of the adult human urethra as claimed in claim 11 wherein the gel is selected from biocompatible delivery system such as combination of fibrinogen and/or thrombin and/or theremo-reversible gelation polymer (TGP) gel and/or collagen and/or chitosan and the like. a. Urethotomy is conducted to relieve the complete urethral stricture using an internal cystoscopic knife. Mainly at 12 O'clock position. b. Stricture area is visualized with scope. c. Foley's catheter of 14 French is passed through urethra and Foley catheter balloon is inflated. d. 6 French Urethroscope is passed through urethra and stricture area is visualized. e. Keeping 6 French scope in urethra, 5 French infant feeding tube is passed. f. Now, cystoscope and catheter along with infant feeding tube is present in urethra. g. With same cystoscopy, stricture area is visualized and tip of infant feeding tube is visualized and directed towards the stricture area at starting of the stricture at bladder side. h. Minimal traction of Foley catheter is given and saline flow is stopped. i. Stricture area to be dried out by suction of saline through infant feeding tube. j. Autologous buccal epithelial cell suspension is implanted at the stricture area with the help of biocompatible delivery system such as combination of fibrinogen and/or thrombin and/or theremo-reversible gelation polymer (TGP) gel and/or collagen and/or chitosan and the like through infant feeding tube at stricture area. Infant feeding tube is little withdrawn till another end of stricture. k. Biocompatible delivery system such as combination of fibrinogen and/or thrombin and/or theremo-reversible: gelation polymer (TGP) gel and/or collagen and/or chitosan and the like is surrounded by Foleys catheter and covers stricture area in a cylindrical form. l. Cystoscope and infant tube is removed, and Foleys catheter is retained in urethra. m. Wait for 6 to 7 minutes holding penis in vertical position and maintain minimal traction on Foleys catheter. n. Penis is secured with strapping on abdomen, also Foleys catheter is strapped, so that Foleys catheter is firm and there is no movement. o. Urine bag is attached to Foleys catheter.

Description

DESCRIPTION OF THE DRAWINGS

[0087] FIG. 1Autologous Buccal Epithelial Cell Culture Process.

[0088] FIG. 2Process steps for the preparation of autologous buccal epithelial ceilsuspension.

[0089] FIG. 3AReal time PCR based autologous buccal epithelial cell characterization (Amplification plot generated after real time PCR using GAPDH gene specific primers).

[0090] FIG. 3BReal time PCR based autologous buccal epithelial cell characterization (Amplification plot generated after real time PCR using CK14+ gene specific primers).

[0091] FIG. 4AFlow cytometry based autologous buccal epithelial cell characterization using CK14.sup.+ surface markers.

[0092] FIG. 4BFlow cytometry based autologous buccal epithelial cell characterization test.

[0093] FIG. 5Result of karyotyping Analysis performed at the autologous buccal epithelial cell manufacturing step.

[0094] FIG. 6Schematic representation with duploject.

[0095] The following examples illustrate preferred embodiments in accordance with the present invention without limiting the scope of the invention.

EXAMPLES

Example 1

[0096] Oral mucosal tissue 1+1.5 cm.sup.2 is harvested from the inner cheek region of an adult human patient with urethral stricture. The harvested tissue is placed in 0.5% enzyme trypsin in calcium and magnesium ion free phosphate buffer saline solution for 16 to 18 hours at 2-8 C. The tissue sample is removed from the solution and washed with DMEM solution. The cellular stratum of the tissue sample is separated with a forcep, minced and filtered to obtain uniform cell suspension of epithelial cells. The cells are suspended in DMEM medium and seeded with in T-25 flask.

[0097] The DMEM medium is replaced every alternate day and when the cell confluency is about 80 to 90% the cellular monolayer is harvested with enzyme trypsin-EDTA. The cellular suspension is subjected to centrifugation and supernatant is discarded. The pellet is mixed with nutrient medium. Appropriate number of cell suspension is filled in transparent V shaped 1 ml vial for transportation to the stricture site.

[0098] Quality control test(s) such as Appearance, Sterility, Mycoplasma, Endotoxin, Cell Counting, Cell Viability, Cell Purity Test, Cell Characterization and Karyotyping Analysis are conducted as mentioned below.

[0099] a) Appearance [0100] Standard; [0101] Red-Colored culture medium (DMEM), which contains mixed precipitated pale-white-colored autologous adult live cultured buccal epithelial cells. This content becomes turbid when shaken.

[0102] b) Sterility Test [0103] Standard: [0104] According to the Sterility Test of Indian Pharmacopoeia biological test methods section 2.2.11, if no evidence of microbial growth is found, the preparation under examination complies with the test for sterility.

[0105] c) Mycoplasma Test (PCR Method) [0106] Standard: [0107] It shall not detect any mycoplasma when tested according to the mentioned test.

[0108] d) Endotoxin Test [0109] Standard: [0110] According to biologics standard and Method D of Bacterial Endotoxins Testing Method under section 2.2.3 of the biological methods in Indian Pharmacopoeia 2007, there should be less than 3 EU/mL endotoxin during testing.

[0111] e) Cell Counting Test [0112] Standard: [0113] It shall be included 2,500,000 cells per 1 vial when tested.

[0114] f) Cell Viability Test [0115] Standard: [0116] Viable cells shall be over 80% of total cell count when tested.

[0117] g) Cell Purity Test [0118] Standard: [0119] Total albumin content shall be below 1.0 g/dL when tested.

[0120] h) Cell Characterization Test: [0121] Standard: [0122] Expression of positive stained cells for CD36.sup.+/CK5.sup.+/CK14.sup.+ antibodies shall be above 80% when tested. [0123] Expression of negative stained cells for CD90.sup. antibodies shall be negligible when tested.

[0124] i) Karyotyping of Cells [0125] Standard: [0126] There should be no chromosomal abnormality on Karyogram imaging and analysis.

Example 2: Analytical Data of Autologous Buccal Epithelial Cellsuspension From 10 Different Subjects

[0127]

TABLE-US-00001 Cell Cell Characterization Tissue Stricture Cell Cell purity (CK14.sup.+ Sample Size Size Cell number Viability Endotoxin test expression) No (in cm) (in cm) Density (10.sup.6) (%) (EU/ml) (g/dl) by FACS U1 1 1 2 0.4 ml 4.90 96.27 <0.769 <0.204 98.60% (2.5 10{circumflex over ()}6) U2 1.5 1.sup. 4 0.8 ml 7.11 96.04 <0.600 <0.182 98.80% (5 10{circumflex over ()}6) U3 1 1 1.5 0.3 ml 5.60 94.67 <0.930 <0.205 97.10% (1.87 10{circumflex over ()}6) U4 .sup.1 0.5 1 0.2 ml 4.48 95.03 <0.600 <0.188 96.30% (1.25 10{circumflex over ()}6) U5 1.5 1.5 4 0.8 ml 8.17 95.96 <0.600 <0.189 97.20% (5 10{circumflex over ()}6) U6 1.5 1.sup. 3 0.6 ml 6.00 98.33 <0.600 <0.187 95.00% (3.75 10{circumflex over ()}6) U7 1.5 1.5 4 0.8 ml 8.31 97.62 <0.600 <0.188 95.60% (5 10{circumflex over ()}6) U8 1.5 1.sup. 3 0.6 ml 8.84 98.02 <0.600 <0.196 98.26% (3.75 10{circumflex over ()}6) U9 1 1 2.5 0.5 ml 7.91 98.02 <0.600 <0.208 97.60% (3.12 10{circumflex over ()}6) U10 1.5 1.5 4 0.8 ml 9.80 98.43 <0.600 <0.208 95.88% (5 10{circumflex over ()}6)

[0128] Other Tests [0129] Micro Sterility: negative in all samples [0130] Mycoplasma: negative in all samples [0131] Cell Characterization (CK14.sup.+ expression) by FACS & RT-PCR: demonstrating high levels of expression of CK14.sup.+ shows the high potency of buccal epithelial cells. [0132] Cell Characterization (CD90.sup. expression) by RT-PCR: Negligible in all samples [0133] Karyotypic Analysis: No chromosomal abnormalities in all samples