Pharmaceutical composition of killed cells with substantially retained immunogenicity

Abstract

This invention discloses the process for lyophilization of the treated cells which comprises the use of a solution containing Trehalose along with amphipathic polymer Polyvinylpyrrolidone. Further the invention discloses the process treating cancer cells, freezing, lyophilizing and reconstituting. Immunomodulator treated dead, but intact cancerous cells were recovered, which can subsequently be used for cancer immunotherapy.

Claims

1. A lyophilized composition comprising: killed cancer cells, by treating with Mycobacterium w (Mw), with substantially retained immunogenicity and morphology: trehalose as an intracellular cryo-preservative at 1 to 10% w/v; and, polyvinyl pyrrolidone as an extra cellular cryo-preservative at 0.1 to 5% w/v.

2. The composition as claimed in claim 1 further comprising an adjuvant.

3. The composition as claimed in claim 1, wherein the polyvinyl pyrrolidone has a molecular weight ranging from 30 to 50 kilo Daltons.

4. The composition as claimed in claim 2, wherein the adjuvant is selected from the group consisting of oils, aluminium salts, virosomes, dead whole organisms, extracts of microbes and combination thereof.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1: Determination of Cellular Size and Granularity of lyophilized cells. Forward Scatter measures Cellular Size and Side Scatter measures the amount of granularity.

(2) FIGS. 2 A & B: Determination of Cellular Integrity using a cell membrane lyophilic stain PKH126 dye.

(3) FIG. 3: Lyophilized cells not stained with the PKH26 dye for determining background fluorescence.

(4) FIG. 4: Cells with intact cell membrane and Nuclear Envelope

(5) FIG. 5: Immunogenicity of lyophilized and then reconstituted cells

(6) FIG. 6: Surface marker staining under fluorescence microscope

DESCRIPTION OF THE INVENTION

(7) Surprisingly it has been found that a stable composition of killed cells with substantially retained immunogenic properties can be prepared comprising of killed cells, at least one intracellular cryo preservative at least one extra cellular cryo preservative and excipients.

(8) The intracellular cryo preservative is selected from carbohydrates such as monosaccharides and diasaccharides. Surprisingly it has been found that trehalose as intracellular cryo preservative for maximizing intact cell recovery yields highest cell recovery. The carbohydrate as an intracellular cryo preservative used is between 1 to 10% W/V concentration.

(9) The extra cellular cryo preservative is selected from amphipathic substances such as Hydroxyethylstarch (HES), Dextran and Polyvinylpyrrolidone (PVP) and ploysorbates.

(10) Surprisingly the amphiphathic polymer Polyvinylpyrrolidone (PVP) has been found to be improving intact cell recovery as extra cellular cryo preservative. The PVP used as an extra cellular cryo preservative is between 0.1 to 5% W/V concentration. The molecular weight of PVP used is in the range 30 to 50 kilo Daltons.

(11) Surprisingly the amphiphathic polymer Hydroxyethylstarch (HES) has been found to be improving intact cell recovery as extra cellular cryo preservative. The HES used as an extra cellular cryo preservative is between 0.1 to 5% W/V concentration. The molecular weight of HES used is in the range 30 to 50 kilo Daltons.

(12) Surprisingly the amphiphathic polymer Dextran has been found to be improving intact cell recovery as extra cellular cryo preservative. The dextran used as an extra cellular cryo preservative is between 0.1 to 5% W/V concentration. The molecular weight of dextran used is preferably in the range of 30 to 50 kilo Daltons.

(13) Surprisingly the amphiphathic polymer polysorbate has been found to be improving intact cell recovery as extra cellular cryo preservative. The polysorbate used as an extra cellular cryo preservative is between 0.1 to 5% W/V concentration. The amphiphathic polymer polysorbate used as extra cellular cryo preservative is selected from Polysorbate 20, Polysorbate 40, Polysorbate 60, Polysorbate 65, Polysorbate 80 preferably Polysorbate 80

(14) In accordance with the Invention the process of preparing a stable composition of killed cells with substantially retained immunogenic properties is in following steps

(15) 1. Treating killed cells with Intracellular cryo-preservatives.

(16) 2. Treating killed cells with Extracellular cryo-preservatives.

(17) 3. Snap chilling in to frozen formulation prior to lyophilisation.

(18) 4. Lyophilisation of treated killed cells.

(19) 5. Reconstitution of lyophilates (of killed cells).

(20) 6. Assessing preservation of cellular integrity.

(21) 7. Assessing effectiveness of preservation method.

(22) 8. Evaluating preservation of antigenic/immunogenic properties.

(23) The lyophilisation solution buffered at pH of 7.4 by Dulbecco's Phosphate Buffered Saline (DPBS—without Calcium Chloride and Magnesium Chloride), the Components of which were Potassium Phosphate Monobasic (0.20 g/L); Potassium Chloride (0.20 g/L); Sodium Chloride (8.00 g/L) and Anhydrous Sodium Phosphate Dibasic (1.15 g/L). The lyophilisation buffer contains a final concentration of about 5% weight by volume of a non reducing disaccharide, preferably Trehalose and a final concentration of about 1% weight by volume of a polymer, preferably Polyvinylpyrrolidone (Mol. Weight. 44K).

(24) The morphological preservation, integrity and retention of immunogenicity even on prolonged storage of killed cell composition were analyzed as follows 1. Morphological profiling (Size):—Flow Cytometry. PKH26 for integrity of ell membrane. Hematoxylin and Eosin staining for cytoplasmic and nuclear membrane integrity 2. Physiochemical Profiling:—Confirming Cell death uses TOPRO3 Iodide Dye. 3. Granularity assay:—FACS FSC/SSC 4. Cytogenetic analysis 5. DNA Profiling: FACS DNA content Analysis. 6. DNA Extraction and agarose gel electrophoresis to assess DNA integrity 7. Immunophenotyping:—HLADR molecules 8. Immunological Profiling:—CD4.sup.+. Cytotoxic evaluation of CD8.sup.+ and NK Cells with cyto-toxic determinants: Perforin and Granzyme. 9. Functional Assay:—FACS Effector Function Assay using PKH26 and TOPRO3 iodide to identify/confirm and enumerate dead Target cells. Quantification of IFNγ and IL-2 producing splenocytes. Lymphocyte Proliferation assay Using PKH 26.

Example 1: Extracellular Cryopreservative

(25) 10.sup.7 cells/ml of cancer cells were treated with Mycobacterium w (Mw). Percentage cell death was determined employing Trypan blue dye exclusion principle. Cells were pelleted by centrifugation at 1500 rpm for 10 min. The Supernatant was discarded and control Pellet was resuspended in DPBS. The total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted. Treatment group pellet was re-suspended in Lyophilisation buffer solution containing 1% PVP w/v. Each aliquot was subjected to snap chilling in Liquid nitrogen (below −100° C.) followed by lyophilisation. Treatment group percent intact cell recovery, estimated after reconstitution of lyophiliates, was 19.125±3.275

Example 2: Intracellular Cryopreservative

(26) 10.sup.7 cells/ml of cancer cells were taken and treated with Mw. Percentage cell death was determined employing Trypan blue dye exclusion principle. Cells were pelleted by centrifugation at 1500 rpm for 10 min. Supernatant was discarded and control group cell pellet was resuspended in DPBS. Total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted. Treatment group cell pellet was resuspended in Lyophilisation buffer solution containing 5% Trehalose w/v. Each aliquot was subjected to snap chilling in Liquid nitrogen (below −100° C.) followed by lyophilisation. Treatment group percent intact cell recovery upon reconstitution of lyophilate in DPBS, estimated using hemocytometer, was 15.27±0.64.

Example 3: Surfactant Polymers for Cryopreservative Activity

(27) 10.sup.7 cells/ml of cancer cells were taken and treated with Mw. Percent cell viability was determined using trypan blue exclusion assay. Cells were pelleted by centrifugation at 1500 rpm for 10 min. Supernatant was discarded and control group cell pellet resuspended in DPBS. Total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted. Treatment group cell pellet was resuspended in Lyophilisation buffer containing 0.05% Polysorbate 80 v/v. Each aliquot was subjected to snap chilling in Liquid nitrogen (below −100° C.) followed by lyophilisation. Treatment group percent intact cell recovery upon reconstitution of lyophilate in DPBS, estimated using hemocytometer, was about 0.9.

Example 4: Extracellular Cryopreservative Treatment Followed by Intracellular Cryopreservative Treatment

(28) 10.sup.7 cells/ml of cancer cells were treated with Mw. Percentage cell death was determined employing Trypan blue dye exclusion principle. Cells were pelleted by centrifugation at 1500 rpm for 10 min. Supernatant was discarded and control group cell pellet resuspended in DPBS. Total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted. Treatment group cell pellet was resuspended in PVP at final concentration of 1% w/v (in DPBS). Cell suspension was incubated at 37° C. for 15 min. subsequently, trehalose was added at final concentration of 5% w/v (in DPBS) followed by incubation at 37° C. for 15 min. Total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted. Prior to lyophilisation, each aliquot was subjected to snap chilling in Liquid nitrogen (below −100° C.). Treatment group Percent intact cell recovery, upon reconstitution of lyophilate in DPBS was 28.08±3.63

Example 5: Intracellular Cryopreservative Treatment Followed by Extracellular Cryopreservative Treatment

(29) 10.sup.7 cells/ml of cancer cells were treated with Mw. Percent cell viability was determined using trypan blue exclusion assay. Cells were pelleted by centrifugation at 1500 rpm for 10 min. Supernatant was discarded and control cell pellet resuspended in DPBS. Total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted. Treatment group cell pellet was resuspended in trehalose at final concentration of 5% w/v (in DPBS) followed by incubation at 37° C. for 15 min. Subsequently PVP was added at final concentration of 1% w/v and cell suspension was incubated at 37° C. for 15 min. Total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted. Prior to lyophilisation, all aliquots were subjected to snap chilling in Liquid nitrogen (below −100° C.). Treatment group percent intact cell recovery was 55.61±4.35.

Example 6: Intracellular Cryopreservative Followed by Extracellular Cryopreservative

(30) 10.sup.7 cells/ml of cancer cells were treated with Mw. Percentage cell death was determined employing Trypan blue dye exclusion principle. Cells were pelleted by centrifugation at 1500 rpm for 10 min. Supernatant was discarded and control group cell pellet resuspended in DPBS. Total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted. Treatment group cell pellet was resuspended in dextrose at final concentration of 5% w/v (in DPBS) followed by incubation at 37° C. for 15 min. Subsequently HES was added at final concentration of 1% w/v and cell suspension was incubated at 37° C. for 15 min. Total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted. Prior to lyophilisation, all aliquots were subjected to snap chilling in Liquid nitrogen (below −100° C.). Post lyophilisation, treatment group percent intact cell recovery was ˜54.88, but cellular morphology was disturbed.

Example 7: Intracellular Cryopreservative Followed by Extracellular Cryopreservative

(31) 10.sup.7 cells/ml of cancer cells were treated with Mw. Percent cell viability was determined using trypan blue exclusion assay. Cells were pelleted by centrifugation at 1500 rpm for 10 min. Supernatant was discarded and control group cell pellet was resuspended in DPBS. Total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted. Treatment group cell pellet was resuspended in Glycerol at final concentration of 5% v/v (in DPBS) followed by incubation for 15 min. at 37° C. Subsequently HES was added at final concentration of 1% w/v and cell suspension was incubated at 37° C. for 15 min. Total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted prior to lyophilisation. All aliquots were subjected to snap chilling in Liquid nitrogen (below −100° C.). Post lyophilisation, treatment group percent intact cell recovery was ˜46.19, albeit cellular morphology was disturbed.

Example 8: Intracellular Cryopreservative Followed by Extracellular Cryopreservative

(32) 10.sup.7 cells/ml of cancer cells were treated with Mw. Percentage cell death was determined employing Trypan blue dye exclusion principle. Cells were pelleted by centrifugation at 1500 rpm for 10 min. Supernatant was discarded and control group cell pellet was resuspended in DPBS. Total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted. Treatment group cell pellet was resuspended in Sucrose at final concentration of 5% w/v (in DPBS) followed by incubation for 15 min. at 37° C. Subsequently HES was added at final concentration of 1% w/v and cell suspension was incubated at 37° C. for 15 min. Total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted prior to lyophilisation. All aliquots were subjected to snap chilling in Liquid nitrogen (below −100° C.). Post lyophilisation, treatment group percent intact cell recovery was ˜47.25, but cellular morphology was disturbed.

Example 9: Extracellular Cryopreservative Followed by Intracellular Cryopreservative with Additives

(33) 10.sup.7 cells/ml of cancer cells were treated with Mw. Percent cell viability was determined using trypan blue exclusion assay. Cells were pelleted by centrifugation at 1500 rpm for 10 min. Supernatant was discarded and control group cell pellet was resuspended in DPBS. Total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted. Treatment group cell pellet was resuspended in PVP at final concentration of 1% w/v (in DPBS) followed by incubation for 15 min. at 37° C. Subsequently, trehalose at final concentration of 5% w/v was added, followed by additives (Nicotinic Acid 0.75 mM; Glutamine 0.75 mM; MgCl.sub.2 0.49 mM and Histidine 5 mM) at final concentration of 1%. Cell suspension was incubated at 37° C. for 15 min. Cells in each aliquot were counted prior to lyophilisation. All aliquots were subjected to snap chilling in Liquid nitrogen (below −100° C.). Post lyophilisation, treatment group percent intact cell recovery was ˜11.0

Example 10: Intracellular Cryopreservative Followed by Extracellular Cryopreservative with Additives

(34) 10.sup.7 cells/ml of cancer cells were treated with Mw. Percentage cell death was determined employing Trypan blue dye exclusion principle. Cells were pelleted by centrifugation at 1500 rpm for 10 min. Supernatant was discarded and control group cell pellet was resuspended in DPBS. Total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted. Treatment group cell pellet was resuspended in Trehalose at final concentration of 5% w/v (in DPBS) followed by incubation for 15 min. at 37° C. Subsequently, PVP at final concentration of 1% w/v was added, followed by additives (Nicotinic Acid 0.75 mM; Glutamine 0.75 mM; MgCl.sub.2 0.49 mM and Histidine 5 mM) at final concentration of 1%. Cell suspension was incubated at 37° C. for 15 min. Cells in each aliquot were counted prior to lyophilisation. All aliquots were subjected to snap chilling in Liquid nitrogen. Post lyophilisation, treatment group percent intact cell recovery was ˜48.9

Example 11: Freezing Method

(35) 1×10.sup.7 cells/ml were treated with Mw. Percentage cell death, employing Trypan blue dye exclusion principle, was 60%. 5 aliquots were made and cells were pelleted down by centrifugation at 1500 rpm for 10 min. Of the 5 cell pellets obtained, 1 each resuspended in 100 μl of 50× Trehalose followed by incubation for 15 min. at 37° C. Final volume made upto 1 ml with DPBS. From each aliquot 200 μl distributed in glass vials labeled appropriately and cells in each aliquot were counted. All aliquots were subjected to slow freezing viz. at 8° C. for 1 hr, 4° C. for 2 hr, −20° C. for 4 hr and finally at −−70° C. for 8 hrs. Except for one freezing control, rest all aliquots subjected to lyophilisation for approx 48 hrs. Lyophilates reconstituted with 200 μl of DPBS and total no of intact cells counted using haemocytometer. Freezing control yielded 13% intact cells while lyophilisation resulted in 2% recovery.

Example 12: Freezing Method

(36) 1×10.sup.7 cells/ml were treated with Mw. Percentage cell death, employing Trypan blue dye exclusion principle, was 60%. 5 aliquots were made and cells were pelleted down by centrifugation at 1500 rpm for 10 min. Of the 5 cell pellets obtained 1 each resuspended in 100 μl of 10× PVP followed by incubation for 15 min. at 37° C. Final volume made upto 1 ml with DPBS. From each aliquot 200 μl distributed in glass vials labeled appropriately and cells in each aliquot were counted. All aliquots were subjected to slow freezing viz. at 8° C. for 1 hr, 4° C. for 2 hr, −20° C. for 4 hr and finally at −70° C. for 8 hrs. Except for one freezing control, rest all aliquots subjected to lyophilisation for approx 48 hrs. Lyophiliates reconstituted with 200 μl of DPBS and total no of intact cells counted using haemocytometer. Freezing control yielded 32% intact cells while lyophilisation resulted in 5% recovery.

Example 13: Freezing Method

(37) 1×10.sup.7 cells/ml were treated with Mw. Percentage cell death, employing Trypan blue dye exclusion principle was 60%. 5 aliquots were made and cells were pelleted down by centrifugation at 1500 rpm for 10 min. Of the 5 cell pellets obtained 1 each resuspended in 100 μl of 10× Dextran Sulfate followed by incubation for 15 min. at 37° C. Final volume made upto 1 ml with DPBS. From each aliquot 200 μl distributed in glass vials labeled appropriately and cells in each aliquot were counted. All aliquots were subjected to slow freezing viz. at 8° C. for 1 hr, 4° C. for 2 hr, −20° C. for 4 hr and finally at −70° C. for 8 hrs. Except for one freezing control, rest all aliquots subjected to lyophilisation for approx 48 hrs. lyophilized cell pellets reconstituted in 200 μl of DPBS and total no of intact cells counted using haemocytometer. Freezing control yielded 5% intact cells while no intact cell was observed after lyophilisation.

(38) Though several intra-cellular and extra-cellular cryopreservatives were capable of preserving the morphology and intactness of killed cells, trehalose and PVP appear advantageous over others.

Example 14

(39) 1×10.sup.7 B16F1 cells/ml treated with Mw. Percentage Cell death, employing Trypan blue dye exclusion principle was 25%. Cells were pelleted by centrifugation at 1500 rpm for 10 min. Supernatant was discarded. Cell pellets were resuspended in either 100 μl of 50× Trehalose or 100 μl 10× PVP and incubated at 37° C. for 15 min. Final volume was made upto 1 ml with DPBS. Five aliquots of 200 μl each were made and cells in each aliquot were counted prior to lyophilisation. Samples snap freezed in liquid nitrogen. Except one freezing control, rest all samples were subjected to lyophilisation for approx 48 hrs. Lyophilates reconstituted with 200 μl of DPBS and total no of intact cells counted using haemocytometer. Freezing control yielded 39% intact cells while lyophilisation resulted in 9% intact cell recovery. A combination of the two cryopreservatives appears advantageous over either of them alone.

Example 15

(40) HEK-293, at a concentration of 1×10.sup.7 cells/ml, was treated with Mw. Percentage Cell death, employing Trypan blue dye exclusion principle was 80%. Cells pelleted by centrifugation at 1500 rpm for 10 min. Supernatant was discarded. Cell pellets were resuspended in 5 ml of DPBS and total volume was distributed in 5 aliquots of 1 ml each. Cells in each aliquot were counted followed by centrifugation at 1500 rpm for 10 min. Cell pellets were resuspended in 100 μl of 50× Trehalose and incubated at 37° C. for 30 min. 100 μl 10× PVP was added subsequently, and samples again incubated at 37° C. of 30 min. Final volume was made upto 1 ml with DPBS. Total volume was distributed into aliquots of 200 μl each. All aliquots were snap freezed in Liquid Nitrogen and except one freezing control rest all subjected to lyophilisation for approx 48 hrs. Lyophilates reconstituted with 200 μl of DPBS and total no of intact cells counted using haemocytometer. Freezing control yielded 67% intact cells while lyophilisation resulted in 49% intact cell recovery. A combination of the two cryopreservatives appears advantageous over either of them alone.

(41) The addition of PVP to Trehalose leads to a higher recovery of intact cells (˜50%) as opposed to either of them alone or addition of Trehalose to PVP.

Example 16

(42) B16F10 cells, at a concentration of 1×10.sup.7 cells/ml, were treated with Mw. Percent cell death, determined using trypan blue exclusion assay, was 31%. Total volume was distributed into 10 aliquots of 1 ml each and number of cells in each aliquot was counted before processing. Cells pelleted by centrifugation at 1500 rpm for 10 min. Cell pellets were resuspended in 100 μl of 50× Trehalose and incubated at 37° C. for 15 min. 100 μl 10× HES was then added and samples were again incubated at 37° C. for 15 min. Final volume was made upto 1 ml with DPBS. 200 μl was distributed in glass vials labeled appropriately. Samples were snap freezed in Liquid Nitrogen and except one freezing control rest all subjected to lyophilisation for approx. 48 hrs. Lyophilates reconstituted with 200 μl of DPBS and total number of intact cells counted using hemocytometer. Freezing control yielded 95% intact cells while lyophilisation resulted in 70% intact cell recovery but significant amount of cells were clumped together.

(43) Trehalose+PVP seem to be best among all other tested combinations of different cryopreservatives.

Example 17: Assessment of Cellular Size and Granularity

(44) Flow cytometry can provide information about the cellular size and granularity from homogeneous or heterogeneous tissue/cell suspension in medium. Cellular size is measured as the diffracted laser light generated from the cell membrane; and granularity is the measure of the reflected and refracted light that is emitted upon targeting the granules of a cell. Cellular size is measured on the Forward Scatter (FSC) scale of the dotplot; and granularity on the Side Scatter (SSC) scale of the dotplot. Assessment of the lyophilized cells indicate two distinct population, as shown in FIG. 1. Population P1 appears at a lower FSC and SSC population that population P2, suggesting that population P2 are larger in size and posses more granules compared to population P1.

Example 18: Determination of Cellular Integrity

(45) Lyophilized cells were stained with cell membrane lipid binding molecule PKH26. PKH26 is excited by the blue laser and absorbs light at 551 nm and emits light at 567 nm. FIG. 2 shows that both P1 and P2 population uptake the PKH26 dye. Lyophilized cells stained with the PKH26 dye, 90.7% of P1 population and 96.3% of P2 population are PKH26+ve.

Example 19: Determination of Cell Death

(46) Cell death was evaluated by Propodium Iodide (PI) dye, which penetrates into cells with compromised cell membrane. FIG. 3 shows that both P1 and P2 uptake the PI dye, suggesting that both the population are dead. Lyophilized cells stained with PI dye. 78.3% of P1 population and 97.8% of P2 population are PI+ve.

Example 20: Cells with Intact Cell Membrane and Nuclear Envelope

(47) The formulation was stained with Hematoxylin and Eosin to assess cytoplasmic and nuclear membrane integrity. Intact Cells with intact nucleus were observed (FIG. 4).

Example 21

(48) MiaPaCa 2 cells, at a concentration of 1×10.sup.7 cells/ml, were treated with Mw. Percentage cell death, determined using trypan blue exclusion assay, and was 100%. Total volume was distributed into 2 aliquots of 5 ml each and number of cells in each aliquot was counted before processing. Cells pelleted by centrifugation at 1500 rpm for 10 min. Cell pellets were resuspended in 100 μl of 50× Trehalose and incubated at 37° C. for 15 min. 100 μl 10× PVP was added subsequently, and aliquots again incubated at 37° C. of 15 min. 200 μl was distributed in glass vials labeled appropriately and were snap freezed in Liquid Nitrogen. One set of vials were lyophilized for approx. 48 hrs. Lyophilates reconstituted with 200 μl of DPBS and cell suspension was injected in Balb/C mice on day 1 and 21. Non-lyophilized formulated cells were administered in control group on day 1 and 21. On day 28, all mice were sacrificed and the splenocytes were isolated. Interferon gamma ELISPOT was performed to assess immune response. The lyophilized cells showed equal number of cells producing interferon gamma indicating retained immunogenicity, infect little better. (FIG. 5).

Example 22

(49) Killed 10.sup.7 cells/ml of cancer cells were pelleted by centrifugation at 1500 rpm for 10 min. Supernatant was discarded. Total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted. Treatment group cell pellet was re-suspended in trehalose at final concentration of 5% w/v (in DPBS) followed by incubation at 37° C. for 15 min. Subsequently PVP was added at final concentration of 1% w/v and cell suspension was incubated at 37° C. for 15 min. Total volume was distributed into aliquots of 1 ml each and cells in each aliquot were counted. Prior to lyophilisation, all aliquots were subjected to snap freezing in Liquid nitrogen. Cells were reconstituted in DPBS.

(50) A Smear was prepared on a glass slide; air dried and fixed using acetone. Blocking for 1 hour with Blocking Buffer containing 5% BSA, Serum, and 2% Triton-X 100 in PBS. Incubated 1 hour with primary antibody targeted against a cell surface marker was used for detection at 1:100 dilutions. Wash with PBS containing triton. Incubated with Anti mouse IgG-FITC labeled Secondary Ab (1:1000). Wash with PBS containing triton.

(51) The surface proteins are detected under florescent microscope as depicted in FIG. 6. The method can be used for the transport of samples tissue killed or live and later use them for diagnosis or forensic.

(52) Both intra-cellular and extra-cellular cyropreservatives are capable of preserving the morphology of Mw treated cells. PVP and trehalose appear advantageous over both dextran and Polysorbate-80 wherein the cells appeared to be clumped.

(53) A combination of the two cryopreservatives appears advantageous over either of them alone. The addition of PVP to Trehalose leads to a higher recovery of intact cells (approx 50%) as opposed to either of them alone or addition of trehalose to PVP.

(54) The method of preservation by lyophilisation can be used for preserving whole cells vaccine candidates with retained immunogenicity, intact structure and nucleic acid. The method can also be used to preserve cell samples for forensic applications and diagnostic purposes.