PROCESS OF PREPARING CHONDROCYTE CELL SUSPENSION AND ITS USE

Abstract

A process for the preparation of chondrocyte cell suspension and its use in defect site of knee or ankle or shoulder or wrist or elbow or hip of subject.

Claims

1. A process of preparing chondrocyte cell suspension comprising (a) harvesting 40 to 100 mg weight of cartilage tissue from non-weight bearing area of knee of the subject; (b) mincing the tissue from (a) followed by digesting with enzyme(s) for the isolation of chondrocyte cells; (c) mixing chondrocyte cells with nutrient medium, serum and optionally growth factors; (d) optionally seeding to enable cell multiplication until P2 stage to obtain not less than 48 million cells within four weeks; (e) centrifuging, discarding the supernatant; (f) mixing with nutrient medium; (g) analyzing and characterizing the chondrocyte cell suspension; (h) filling the characterized chondrocyte cell suspension in transparent V shaped 1 ml vials; and optionally transporting to the same subject as in (a).

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

3. A process of preparing chondrocyte cell suspension as claimed in claim 1 wherein the enzyme is selected from trypsin-EDTA, Collagenase and the like.

4. A process of preparing chondrocyte cell suspension as claimed in claim 1 wherein the nutrient medium is selected from IMDM, EMEM, DMEM and the like.

5. A process of preparing chondrocyte cell suspension as claimed in claim 1 wherein the growth factors are selected from IGF, TGF, FGF and the like.

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

7. A process of preparing chondrocyte cell suspension as claimed in claim 1 wherein the transportation is at 2 to 8 degree centigrade.

8. A process of preparing chondrocyte 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 chondrocyte cell suspension as claimed in claim 9 wherein cell characterization is by analyzing chondrocytes CD44.sup.+ and CD151.sup.+ marker expressions.

10. A process of preparing chondrocyte cell suspension as claimed in claim 1 further comprising optionally mixing with gel while implanting the chondrocyte cell suspension into the defect site of the knee of the subject by using arthroscopy or mini arthrotomy.

11. A process of preparing chondrocyte cell suspension as claimed in claim 11 wherein the defect size ranges from 1 to 20 cm.sup.2.

12. A process of preparing chondrocyte cell suspension as claimed in claim 11 wherein the gel is selected from fibrin, thrombin, thermoreversible gel and the like.

13. A process of preparing chondrocyte cell suspension as claimed in claim 11 wherein when gel is used implantation is carried out using Y-shaped canula comprising a blunt needle.

14. A method of implanting chondrocyte cell suspension for autologous chondrocyte transplantation comprising (i) drawing 1 ml of nutrient media from vial 1, mixing with fibrin (concentration ranging from 72 to 110 mg) in vial 2, mixing and aspirating the contents into syringe A; (ii) drawing 1 ml of nutrient media from vial 1, adding to thrombin (concentration of 500 IU/ml) in vial 3 and mixing; (iii) drawing 0.2 ml from vial 3 and injecting into empty vial 4; (iv) subsequently drawing 0.4 ml+0.4 ml from vial(s) of chondrocyte cell suspension, injecting into vial 4 and aspirating the contents into syringe B; (v) placing syringe A and B on the applicator/holder; (vi) fixing Y-shaped dual syringe applicator comprising a blunt needle to the two syringes; and (vii) implanting into the defect site of the subject using arthroscopy or mini arthrotomy.

15. A method of implanting chondrocyte cell suspension as claimed in claim 14 wherein nutrient medium is selected from IMDM, EMEM, DMEM and the like.

16. A method of implanting chondrocyte cell suspension as claimed in claim 14 wherein implantation is carried out using Y-shaped dual syringe applicator comprising a blunt needle.

17. A method of implanting chondrocyte cell suspension as claimed in claim 14 wherein chondrocyte cell suspension is prepared by a process as claimed in claims 1 to 9.

18. A method of implanting chondrocyte cell suspension as claimed in claim 14 wherein the subject is an adult human subject.

19. A method of implanting chondrocyte cell suspension as claimed in claim 14 wherein the defect site is in knee or ankle or shoulder or wrist or elbow or hip of subject.

20. A method of implanting chondrocyte cell suspension as claimed in claim 14 wherein the chondrocyte cell suspension is prepared by a process comprising (a) harvesting 40 to 100 mg weight of cartilage tissue from non-weight bearing area of knee of adult human subject; (b) mincing the tissue from (a) followed by digesting with enzyme(s) for the isolation of chondrocyte cells, wherein the enzyme(s) is selected from trypsin-EDTA, Collagenase and the like; (c) mixing chondrocyte cells with nutrient medium, serum and optionally growth factors, wherein the nutrient medium is selected from IMDM, EMEM, DMEM and the like; (d) optionally seeding to enable cell multiplication until P2 stage to obtain not less than 48 million cells within four weeks, wherein the seeding is done in T-25 flask and/or T-75 and/or T-150 flask and the like; (e) centrifuging, discarding the supernatant; (f) mixing with nutrient medium; (g) analysing and characterizing the chondrocyte cell suspension; wherein analysis performed are Appearance, Sterility, Mycoplasma, Endotoxin, Cell Counting, Cell Viability, Cell Purity Test, Cell Characterization and Karyotyping Analysis; further wherein cell characterization of chondrocytes is analyzed by CD44.sup.+ and CD151.sup.+ marker expressions; (h) filling the characterized chondrocyte cell suspension in transparent V shaped 1 ml vials; and optionally transporting to the same subject as in (a) at 2 to 8 degree centigrade.

Description

DESCRIPTION OF THE DRAWINGS

[0101] FIG. 1 Chondrocyte Cell Suspension Process Flow.

[0102] FIG. 2 Process steps for preparing Chondrocyte Cell Suspension.

[0103] FIG. 3 Graphical Representation of Chondrocyte Cell Suspension at primary culture step & final manufacturing step

[0104] Illustrates results of QC parameters i.e. cell number achieved at Primary Culture & Final Process Step respectively and cell viability & cell characterization at Final Process Step respectively; along with results of biopsy weight, that are mandatory for implantation.

[0105] FIG. 4 Realtime PCR based qualitative detection of gene expression for CAP-1 and AGGERCAN genes RT-PCR allows you to detect slight changes in expression between genes or samples and will allow you to analyse genes with very low expression as well.

[0106] FIG. 5 Live/dead staining of chondrocyte cells using Fluorescence microscopy

[0107] Fluorescence staining of chondrocyte cells is more reliable than the standard method of cell viability calculation by hemocytometer method. Fluorescence staining provides us with a clear image of the viable as well as non-viable cells. It is more reliable because of its high specificity and low expression can also be detected. The standard method, relies on manual cell counting with the chances manipulation errors and human sampling errors which is not ideal for accuracy of viability count.

[0108] FIG. 6 Mixing procedure: Schematic representation with Y-shaped dual syringe applicator comprising a blunt needle

[0109] Depending on the defect area (in cm.sup.2), 2 ml or 4 ml of cell-gel mixture will be prepared. If defect size is between 1 cm.sup.2 to 10 cm.sup.2, then, 2 ml cell-gel mixture(s) shall be prepared. Whereas, if defect size is between 7 cm.sup.2 to 20 cm.sup.2, then, 4 ml cell-gel mixture(s) shall be prepared.

[0110] FIG. 7. Representative images for cartilage tissue biopsy procedure from non-weight bearing area of the knee joint.

[0111] FIG. 8. Representative Images of arthroscopic procedure using Chondrocyte Cell Suspension at defect area of the knee joint.

[0112] FIG. 9. Representative images of mini arthrotomy procedure using Chondrocyte Cell Suspension at defect area of the knee joint.

[0113] FIG. 10. Representative images of arthroscopic procedure using Chondrocyte Cell Suspension at defect area of the ankle joint.

[0114] FIG. 11. Representative images of mini arthrotomy procedure using Chondrocyte Cell Suspension at defect area of the ankle joint.

[0115] FIG. 12. Representative images of mini arthrotomy procedure using Chondrocyte Cell Suspension at defect area of the shoulder joint.

[0116] FIG. 13. Representative images for comparison between pre-op MRI & post-op MRI (T2 mapping) for patients treated with Chondrocyte Cell suspension at defect area of the knee Joint.

[0117] The following examples illustrate preferred embodiments in accordance with the present invention without limiting the scope of the invention.

EXAMPLES

Example 1

[0118] 49 mg cartilage specimen is harvested through arthroscopy from the non-weight bearing area of the medial femoral condyle of damaged knee of adult human. The harvested cartilage tissue is placed in a sterile vial containing HBSS at pH ranging from 7.0 to 7.5 and transported to the cell culture laboratory. The cartilage is washed with buffered solution supplemented with antibiotics weighed and minced into small pieces and washed again with buffered solution.

[0119] The minced cartilage is digested with trypsin and the isolated cells are collected. Cell suspension Is centrifuged at 1300 rpm for 5 minutes. The supernatant is discarded, and the cells are resuspended in medium with serum and FGF, counted with a coulter counter and seeded into 25 cm.sup.2 culture flask.

[0120] The cells are cultured in a CO.sub.2 regulated incubator in a humidified 95% O.sub.2/5% CO.sub.2 atmosphere. The cultures are observed daily by inverted phase-contrast microscopy. The culture medium is replaced 3 times a Week. After primary cultures became confluent, the cells are detached by Trypsin-EDTA solution, centrifuged, counted, re-suspended in DMEM and seeded into T-150 culture flasks.

[0121] Medium changes were given on alternate day to feed the cell and when the cellular confluency reaches around 80-90% the cellular monolayer is harvested with help of enzyme may be Trypsin-EDTA. Cellular suspension was subjected to centrifugation and supernatant was discarded. Pellet was mixed with DMEM and cell count may be taken by the coulter counter.

[0122] Cellular viability measured by the trypan blue dye excluding test. Cellular viability above 80% is suitable for the implantation purpose. Quality control tests like Appearance, Sterility, Mycoplasma, Endotoxin, Cell Counting, Cell Viability, Cell Purity Test, Cell Characterization and Karyotyping Analysis are performed before releasing of the cells for the implantation.

[0123] For the characterization of chondrocytes, CD44 and CD151 marker expressions are tested by flowcytometry. In flowcytometry cells were mixed with fluorescence tagged antibodies and incubated for 30 minutes in a dark room at room temperature. The cells are then washed twice with FACS Flow Solution. The sample is centrifuged, and supernatant is discarded. The sample is run through the flowcytometry and readings recorded.

[0124] The cells passing the specified limits are suspended in DMEM, aseptically filled in transparent V shaped vials for its use.

Example 2: Analysis of Chondrocyte Cell Suspension to be Used for Knee Defects

[0125]

TABLE-US-00001 Cell Cell Cell Characterization Number Defect Cell No. Viability Cell No. Viability (CD44.sup.+ and CD of Days Sample size Weight of at PC Step at PC Step at FP Step at FP Step 151.sup.+) at FP Step for Cell No. (cm.sup.2) Biopsy (10.sup.5) (%) (10.sup.6) (%) (%) Culture 1. 7 49 1.28 100 49.04 98.74 96.42 28 2. 9 59 1.56 100 48.56 98.8 97.21 26 3. 10.8 55 1.45 100 49.58 97.82 95.77 28 4. 7.2 45 1.12 100 48.50 97.32 97.74 29 5. 9 52 1.36 100 49.20 97.15 96.41 27 6. 12 60 1.67 100 50.20 98.30 98.11 28 7. 8 51 1.30 100 48.35 98.14 95.33 29 8. 12 50 1.25 100 48.75 98.12 94.46 28 9. 18 56 1.48 100 49.23 98.80 96.88 26 10. 16 57 1.53 100 49.25 97.95 98.10 27 PC: Primary culture step FP: Final Process step Results negative for Mycoplasma and endotoxin tests

[0126] Table represents the efficiency of the process of preparing chondrocyte cell suspension with cartilage tissue of weighing approximately 40 mg to 60 mg and producing NLT 48 million cells within 4 weeks of cell culture period, characterized cells sufficient enough to cover cartilage defect 1 cm.sup.2 to 20 cm.sup.2 in knee joint.

Example 3: Analysts of Chondrocyte Cell Suspension after Culturing Cartilage Loose Fragments

[0127]

TABLE-US-00002 Weight of Cell Cartilage Cell Cell Characterization Fragment Cell No. Viability Cell No. Viability (CD44.sup.+ and CD Loose Body at PC Step at PC Step at FP Step at FP Step 151.sup.+) at FP Step Sr. No (mg) (10.sup.5) (%) (10.sup.6) (%) (%) Sample 1 443.30 0.60 100 54.28 98.88 95.71 (CRM LB 01) Sample 2 414.75 0.63 100 51.81 98.74 90.10 (CRM LB 02) Sample 3 146.80 0.24 100 54.50 99.43 97.22 (CRM LB 03) Sample 4 200.00 0.45 100 48.87 98.31 84.55 (CRM LB 04) Sample 5 122.70 0.28 100 52.70 96.33 91.23 (CRM LB 05) Sample 6 226.50 0.39 100 53.74 93.43 87.89 (CRM LB 06) Sample 7 610.00 0.61 100 49.80 93.14 85.56 (CRM LB 7) Sample 8 250.50 0.47 100 59.46 96.52 99.90 (CRM LB 8) Sample 9 105.30 0.29 100 51.59 96.30 91.50 (CRM LB 9) Table represents the efficiency of the process of preparing chondrocyte cell suspension with cartilage tissue of weighing approximately >100 mg and producing NLT 48 million cells within 4 weeks of cell culture period, characterized cells sufficient enough to cover cartilage defect 1 cm.sup.2 to 20 cm.sup.2 in knee joint.

Example 4: Analysis of Chondrocyte Cell Suspension to be Used for Ankle Defects

[0128]

TABLE-US-00003 Cell Cell Cell Characterization Number Defect Weight of Cell No. Viability Cell No. Viability (CD44.sup.+ and CD of Days Sample size Biopsy at PC Step at PC Step at FP Step at FP Step 151.sup.+) at FP Step for Cell No. (cm.sup.2) (mg) (10.sup.6) (%) (10.sup.6) (%) (%) Culture 1. 3.9 52 0.15 100 48.85 98.21 97.42 29 Table represents the efficiency of the process of preparing chondrocyte cell suspension with cartilage tissue of weighing approximately 52 mg and producing 48.85 million cells within 4 weeks of cell culture period, characterized cells sufficient enough to cover cartilage defect 3.9 cm.sup.2 in ankle joint.

Example 5: Analysis of Chondrocyte Cell Suspension to be Used for Shoulder Defects

[0129]

TABLE-US-00004 Cell Cell Cell Characterization Number Defect Weight of Cell No. Viability Cell No. Viability (CD44+ and CD of Days Sample size Biopsy at PC Stage at PC Stage at FP Stage at FP Stage 151+) at FP stage for Cell No. (cm.sup.2) (mg) (10.sup.6) (%) (10.sup.6) (%) (%) Culture 1. 20 53 1.4 100 48.45 97.34 96.28 28 Table represents the efficiency of the process of preparing chondrocyte cell suspension with cartilage tissue of weighing approximately 53 mg and producing 48.45 million cells within 4 weeks of cell culture period, characterized cells sufficient enough to cover cartilage defect 20 cm.sup.2 in shoulder joint.

Example 6

[0130] Phase III Clinical Trial:

[0131] A Prospective, Open-label, Multicentric Study to Assess the Safety and Efficacy of Autologous Chondrocyte cell suspension in Subjects with Articular Cartilage Defects of the Articulating Joint(s).

[0132] Objectives and Purpose:

[0133] Primary Objective:

[0134] To assess the safety of the Autologous Cultured Chondrocytes implantation in articular cartilage defects of the articulating joint(s).

[0135] Secondary Objective:

[0136] To evaluate the efficacy of Autologous Cultured Chondrocytes implantation in articular cartilage defects of the articulating joint(s).

[0137] Number of Patients:

[0138] 14 patients were enrolled in the study. 14 Patients completed the study.

[0139] Product Detail:

[0140] Autologous Cultured Chondrocytes vial (0.4 mL)

[0141] Appearance:

[0142] Colourless transparent vial product, which contains mixed precipitated pale-white-coloured autologous adult live cultured chondrocytes and red coloured fluid. This fluid becomes turbid when shaken.

[0143] Study Duration:

[0144] Total duration of each subject in the study was approximately 28 weeks.

[0145] Total enrolment duration: Approximately 16 weeks

[0146] Total study duration: Approximately 44 weeks

[0147] Criteria for Evaluation:

[0148] The safety endpoints are: [0149] Incidence of adverse events (AEs) related to therapy

[0150] The efficacy endpoints are: [0151] Change in International Knee Documentation Committee (IKDC) (subjective and objective) at visit 7 relative to visit 1. [0152] Changes in MRI (Not less than 1.5T MRI) findings. [0153] Change in Visual Analogue Scale (VAS) score at visit 7 relative to visit 1

[0154] Efficacy Evaluation [0155] Efficacy evaluation was done from IKDC (International Knee [0156] Documentation committee) score and VAS (Visual Analogue Scale)

[0157] Efficacy Evaluation

[0158] a. Change in International Knee Documentation Committee Score at Visit 07 from Baseline Visit. [0159] Details of International Knee Documentation Committee score (IKDC Score is positive when it increases)

TABLE-US-00005 Total IKDC Total IKDC Percentage Score at Score at Changes Condition Site ID Patient ID Baseline Visit 07 Difference (%) (Effect) C1 1 31.0 85.0 54.0 174.19 Positive C1 3 26.4 50.6 24.2 91.67 Positive C1 4 20.7 54.0 33.3 160.87 Positive C1 6 20.7 64.4 43.7 211.11 Positive C2 1 39.1 71.3 32.2 82.35 Positive C2 2 40.2 72.4 32.2 80.10 Positive C2 4 28.7 54.0 25.3 88.15 Positive C2 5 35.6 41.4 5.8 16.29 Positive C2 6 34.5 64.4 29.9 86.67 Positive C4 1 46.0 77.0 31.0 67.39 Positive C4 4 29.9 78.2 48.3 161.54 Positive C4 6 20.7 74.7 54.0 260.87 Positive C4 7 50.6 79.3 28.7 56.72 Positive C4 8 35.6 78.2 42.6 119.66 Positive Total Mean 32.84 67.49 34.66 118.40 NA Standard Deviation 9.25 13.03 12.99 NA NA

[0160] Conclusion:

[0161] Over all change in IKDC score between baseline and visit 7 was noted as 118.40%, so it was concluded that there is highly statistical significant mean difference between baseline and visit 7. Out of 14 patients, all patients had shown remarkable improvement in MRI for the regeneration of articular cartilage at the defect site, the defect was filled completely with the new articular cartilage compared to baseline, recording were suggestive of complete repaired of defected area and shown continuity with the surrounding articular cartilage with no fissure formation, no cleft formation or any other irregularities in surrounding tissues.

[0162] b. Pain Relief as Per Visual Analogue Score (VAS) at Visit 07 from Baseline Visit [0163] Details of Visual Analogue Scale score VAS Score is positive when it decreases)

TABLE-US-00006 Total VAS Total VAS Percentage score at score at Changes Condition Site ID Patient ID Baseline Visit 07 Difference (%) (Effect) C1 1 80 10 70 87.50 Positive C1 3 60 10 50 83.33 Positive C1 4 100 0 100 100.00 Positive C1 6 90 10 80 88.89 Positive C2 1 50 18 32 64.00 Positive C2 2 50 30 20 40.00 Positive C2 4 85 20 65 76.47 Positive C2 5 72 70 2 2.78 Positive C2 6 81 20 61 75.31 Positive C4 1 70 10 60 85.71 Positive C4 4 70 10 60 85.71 Positive C4 6 60 10 50 83.33 Positive C4 7 70 10 60 85.71 Positive C4 8 70 5 65 92.86 Positive Total Mean 72.00 16.64 55.36 75.12 NA Standard Deviation 14.40 17.03 24.49 NA NA

[0164] Conclusion:

[0165] Over all change in VAS score between baseline and visit 7 was noted as 75.12%, so it was concluded that there is highly statistical significant mean difference between baseline and visit 7. Out of 14 patients, all patients had shown improvement in VAS score and International Knee Documentation Committee score at visit 7 (24 weeks post Implant) compared with baseline scores.

[0166] Safety Evaluation

[0167] No Adverse event nor Serious Adverse Events were noticed during study out of all 14 subjects during the safety evaluation period suggestive of Autologous Adult Live Cultured Chondrocytes implantation is safe treatment option in patients with defect in articular cartilage of joints.