DESC:FIELD OF THE INVENTION
The present invention relates to a method for freezing high cell density peripheral blood mononuclear cells (PBMC), more particularly NK cell enriched PBMC’s wherein the said cells are isolated from healthy human blood, characterized by high cell density and prolonged viability.
BACK GROUND OF THE INVENTION
Peripheral blood mononuclear cells (PBMCs) are immune cells and include lymphocytes (T cells, B cells and NK cells), monocytes and dendritic cells that form the primary components of an immune system. Apart from being a critical constituent of the immune system, PBMCs find wide and varied application in treatment and diagnosis of disease, to study immune responses to a drug or an antigen, and assessment of T-cell responses. Recently PBMCs have been used to study gene expression profile of acute ischemic stroke (Moore et. al., Circulation. 2005 Jan 18; 111(2):212-21). They also specifically find application in cancer immunotherapy as anti-tumor effector cells (Heo et. al., Cryobiology. 15 2009 Apr; 58(2):203-9).
Most commonly, PBMCs are used as effector cells in determining antibody dependent cell-mediated cytotoxicity (ADCC) of several therapeutic antibodies. Conventionally, they have been freshly extracted from healthy human blood and purified using ficoll gradient for immediate application. However, fresh extraction requires frequent withdrawal of blood from human volunteer(s) which becomes difficult on a routine basis. To address the challenges of such frequent withdrawal of blood, patent application IN861/CHE/2014 describes a method of culturing freshly isolated PBMCs to produce high cell density PBMCs, wherein the cells preserve their functional activity for up to 25 days. Due to such limited period of activity of cultured high cell density PBMC’s there is a need to have a method that preserve PBMCs cells for longer period of time without losing their viability and functionality.
Cryopreservation is one of the methods available for long term storage of PBMCs and its subsequent use. Further, cryopreserved peripheral blood mononuclear cells are useful in clinical trials that have “low end-point frequencies because they allow the immunologic assays to be performed after the conclusion of the studies, when all end points have already been identified.”
Cryopreservation is a process of “slow rate of cooling”, as a result of which cells tend to remain in equilibrium with the external solution. As the rate of cooling increases with the drop in temperature and achieves a super cool state, there is very little time left for cytoplasmic water to move out of the cells resulting into “ice crystal” formation. These ice crystals lead to physical stress like membrane lipid phase changes, depolymerization of the cytoskeleton, osmotic shrinkage, mechanical damage and denaturation of the proteins. It is the cellular response to stress that will determine the survival and revival of cells.
However, cryopreservation of high cell density PBMCs pose a challenge which impact functionality of the cells as density is very high in any vial. Hence, during rapid cooling the formation of “ice crystals” will be in close proximity to the shrunk cells which can lead to damage of cells.
In order to overcome and reduce the physical stress that a cell undergoes during solidification at temperatures of -80ºC and -136ºC, cryoprotectants or cryoprotective agents (CPA) are available. These cryoprotective agents maintain a conducive normo-osmotic environment during freezing that prevents cells from getting shrunken beyond specific point (eutectic point) and also prevent cells from osmotic shock during rapid thawing at 37ºC.
Cryoprotective agents (CPA) include alcohols (glycerol, methanol, 2-propanol), sugars (sucrose, xylitol, dextrose, trehalose), polyethylene glycols (ethylene glycol, PEG 200, PEG 2000, PEG 20000), glycols [dimethyl sulfoxide (DMSO), 2-methyl-2,4-pentanediol (MPD)], and salt (NaCl) and mixture thereof.
Depending upon the rate of penetration into the cells, CPAs are classified as “Permeable CPAs” and “Impermeable CPAs”. The permeability of some of the CPAs also depends on temperature and cell type. Cell permeable CPAs include DMSO, glycerol, sugars such as trehalose, and low molecular weight polymers. Cell impermeable CPAs include high molecular weight CPAs.
Sub-optimal cryopreservation results in a significant decrease of cell viability and number, and may also cause alterations of the cellular phenotype and a reduction of the immunogenic response to specific antigens (Costantini et al., 2003).
Hence, it is very critical to select appropriate cryoprotectants to achieve successful cryopreservation in which viability and function of the cells are not effected during cryopreservation and its revival.
Hence, the primary objective of the invention is to provide an effective cryopreservation method for high cell density PBMCs wherein the said cells does not lose their effector function during cryopreservation and its revival.
Another objective of the invention is to produce cryopreserved PBMCs wherein the said cells elucidate in vitro response equivalent to the cultured PBMCs response.
Yet, another objective of the invention is to provide a method for cryopreservation wherein the cryopreserved high cell density can be thawed and further propagated for a said period of time and be used in functional assays such as ADCC as source effector cells without losing their activity.
SUMMARY OF THE INVENTION
The present invention discloses a method for cryopreservation of cultured high cell density PBMCs, wherein the said method preserves cell survival and activity/function of the cell during cryopreservation as well as rapid thawing at 37?C. Further, the method uses a composition comprising a low molecular weight permeable cryoprotectant and a high molecular weight non- permeable cryoprotectant wherein the method improves post thaw viability of frozen PBMCs. Furthermore, the said cryopreserved cells retain the functionality of the enriched NK cells in the culture containing human PBMCs upon freezing and post thaw. The cells cryopreserved according to the methods of the present invention are revived after thawing and further propagated in a suitable culture medium. The said cells retain their functional activity and is used as source effector cells in ADCC assay.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1: Illustrates a representative dose response curve in ADCC assay of an antibody using cultured cryoproserved PBMC’s as well as unfrozen freshly cultured PBMC’s.
Figure 2: Illustrates comparative assessment between cultured cryopreserved PBMC’s and unfrozen freshly cultured PBMC’s on different days in terms of signal obtained.
Figure 3: Illustrates comparative assessment of relative potency between cultured cryopreserved PBMC’s and unfrozen freshly cultured PBMC’s on different days as source effector cells.
Figure 4: Correlation plot of ADCC activity with % AF content. Four lots of a mAb which had different % Afucosylation were analyzed in ADCC. The Relative ADCC activity and % AF of four lots was plotted on Y and X axis respectively.
DESCRIPTION OF THE INVENTION
The present invention discloses a method for cryopreservation of high cell density PBMCs wherein the said method employs a composition comprising two or more cryoprotectants such that said composition helps in preserving cell survival and cell function during freezing and its subsequent use.
In one embodiment the invention provides a method of performing cryopreservation of high cell density PBMC comprising,
a) adding a freezing mixture comprising about 4 to 6% of hydroxy ethyl starch (HES), Di methyl sulfoxide (DMSO), pooled human serum and growth medium to cultured PBMC
b) cooling the said solution at 1ºC/min in a cooler and storing at -80 ºC.
In yet another embodiment the invention discloses a method, wherein the concentration of HES in freezing mixture is about 4 to 4.5%.
In another embodiment the invention discloses a method, wherein the concentration of DMSO is about 10% (v/v).
In another embodiment the invention discloses a method wherein, the growth media comprises 10% Lymphocyte Growth Medium-3 (LGM3) with 50ng/ml Recombinant human interleukin-2 (rhuIL2).
In another embodiment the invention discloses a method, wherein the concentration of pooled human serum is about 50% (v/v).
In yet another embodiment of the invention, the present invention discloses a method wherein the density of PBMC is in the range of 107 -108 cells.
In another embodiment of the invention, the cells obtained from the said cryopreservation method can be propagated for up to 21 days without losing their viability or activity.
In further embodiment of the invention, the said propagated PBMCs post cryopreservation and thawing are can be used as effector cells in functional assay such as ADCC.
In another embodiment of the invention, the activity of said cryopreserved PBMCs having a biological activity upon thawing and subsequent culturing is comparable to freshly cultured PBMC when used in a functional assay such as ADCC.
In another embodiment, the invention discloses a composition comprising cryopreservation medium for, culturing PBMC wherein the said medium comprises HES, DMSO, pooled human serum and growth medium, and wherein the said PBMC cells are viable and active.
In yet another embodiment, the invention discloses source effector cells such as PBMC’s which retain their activity in ADCC assay for atleast 20 days post freezing, thawing and subsequent culturing.
In yet another embodiment, the invention discloses cryopreserved PBMCs as source effector cells in ADCC assay wherein the functionality of said cells are demonstrated by correlation between afucosylated glycoforms of an antibody in ADCC assay.
In another embodiment the invention discloses, a cryopreservation method for high cell density PBMCs, comprising: suspending PBMCs in a cryopreservation medium, freezing the suspension of the PBMCs in the cryopreservation medium by cooling it to -80°C or below at a cooling rate of 1ºC/min, and storing the frozen suspension of PBMC in the cryopreservation medium; said medium comprising HES, DMSO, polled human serum and growth medium.
The method disclosed herein the invention provides cryopreserved high cell density PBMCs wherein the said cells are propagated further for a period of time upto 20 days after thawing without losing their activity in ADCC.
Certain specific aspects and embodiments of the invention are more fully described by reference to the following examples. However, these examples should not be construed as limiting the scope of the invention in any manner.

EXAMPLES
Prior to the cryopreservation of PBMC’s, first PBMCs were extracted and cultured to achieve high cell density of NK cell enriched population followed by cryopreservation of the obtained high cell density NK cell enriched PBMCs using appropriate cryoprotective agents to use them as source effector cells in ADCC assay and details of the same has given in below examples.
Example 1: Extraction of PBMCs from human blood
Whole human blood was drawn from the veins of a healthy human volunteer under aseptic conditions using a 21g needle and transferred to vacutainers containing Heparin. PBMCs were isolated within one hour of blood collections after diluting it with Mc Coy’s 5A/RPMI 1640 plain medium in the ratio of 1:1. 7 mL of the diluted blood was over layered on 3.5 mL of ficoll in a 15 mL centrifuge tube and centrifuged at 1000 x g and 18-20 °C without brakes for 30 minutes. The buffy coat containing the PBMCs was aspirated and washed 5 three times with Mc Coy’s 5A/RPMI 1640 media in the ratio of 1:1 at 443 xg for 10 minutes with brakes. After the third wash the pellet was resuspended in 3-5 mL of LGM-3TM plain media (Lonza India Pvt. Ltd.,) and the viable cell count was performed using Trypan Blue (Sigma Life Sciences, Sigma-Aldrich) dye exclusion method.
Example 2: Culturing of PBMCs
The isolated PBMCs obtained from example 1, were seeded at a density of 1 x 106 cell/mL in a growth media containing 90 % LGM-3, 10 % heat inactivated human serum (Bioreclamation LLC.,) and 50 ng/mL of recombinant human interleukin-2 (R&D Systems Inc.,) in a T-flask. After attaining the desired confluency, the content of the flask was collected and centrifuged at 443 x g and the pellet so obtained was resuspended in the afore mentioned growth medium. The cell count was performed using Trypan Blue Dye exclusion method.
Example 3: Freezing of PBMC’s
Prior to the cryopreservation of PBMC’s, combination of two different cryoprotectants such as HES and DMSO were selected to be employed in the freezing medium to preserve cell viability and functionality during freezing and its subsequent use. Cultured high cell density NK Cell enriched PBMC’s from example 2 was centrifuged at 443 x g for 10 minutes at room temperature. After centrifugation, supernatant was discarded and the pellet was dislodged by gentle tapping. Further, the pellet was resuspended into 20-25 mL volume of 10% Lymphocyte Growth Medium-3 (LGM3) complete media with 50ng/mL Recombinant human interleukin-2 (rhuIL2) and the resuspended pellet was mixed gently to make a uniform suspension. Freezing mixture containing following composition 4.5% (w/v) HES, 40% (v/v) Growth Media (10% LGM3 with 50ng/mL rhuIL2), 50% (v/v) Pooled Human Serum, 10% (v/v) DMSO was prepared in dark at room temperature.
High cell density NK cell enriched PBMC’s were centrifuged again at 443 x g for 10 minutes. Supernatant was discarded. The prepared freezing mixture was added through the wall of the centrifuge tube of the pellets to attain a density of 2 x107cells to 6 x 107 cells/mL of suspension. Further, 3 x 107cells – 9 x 107 cells/1.5mL suspension aliquots were transferred to 1.8 mL cryo tubes and tubes were placed on a 1?C cooler at -80?C overnight. After placing in a 1?C cooler at -80?C overnight the said content was transferred to a liquid nitrogen container for further use.
Example 4: Thawing and Revival of Frozen human PBMCs and culturing thawed cells for evaluation of Source effector cells.
The effectiveness of cryopreservation of high cell density NK cell enriched PBMCs in a freezing mixture comprising combination of two cryoprotectants such as HES and DMSO on revival, functionality and subsequent propagation of the cells was assessed further. Revival media was prepared for thawing frozen PBMCs and composition of revival media as follows 50% LGM3 media with 50ng/mL rhuIL2. 10 mL of revival media was aliquoted in a 15mL centrifuge tube. Cryopreserved PBMCs containing vials from the liquid nitrogen were placed in a pre-cooled 1?C cooler. The said vials were immediately thawed in a 37?C water bath and the content was transferred to the 15mL centrifuge tube containing 10 mL revival media. Further, the said vials containing cells were centrifuged at 443 x g for 5 minutes. Supernatant was discarded and the pellet was again resuspended in 2 mL -5mL revival media and volume was made upto 10 mL and the content was transferred to T75cm2 flask. Further, the cells were counted using trypan blue dye exclusion technique. The said flask was incubated at 37?C in 5% CO2 microenvironment overnight. Content present in the flask was centrifuged at 443 x g for 5 minutes in the subsequent day. The obtained pellets were resuspended in 10 mL of 10% LGM3 media containing 50ng/ml of rhuIL2 and the content was transferred to T75cm2 flask. The flask was incubated at 37?C in 5%CO2 microenvironment for further growth.
Example 5 Assessment of high cell density NK cell enriched PBMCs as source effector cells using ADCC
Cryopreserved high cell density PBMCs using freezing mixture comprising two different cryoprotectants were thawed and cultured as described in example 4. The cells were evaluated for their functionality as source effector cells using ADCC assay methodology as follows.
Test samples of a therapeutic monoclonal antibody were appropriately diluted in assay medium (90% RPMI/Mc Coys’s M5 + 1% heat inactivated Fetal Bovine Serum) to prepare 8-10 different concentrations. The varying concentration of therapeutic monoclonal antibodies were added to different wells including media control to obtain dose response curve. The target cells were seeded to different wells at a density of 5 x 104 cells/wells except for the media control (MC) and the effector control (EC) wells. The plate was incubated under 5 ± 0.5% CO2 at 37 °C ± 1 °C for 1 h. PBMCs that were 9 days to 25 days old in culture as well as cultured cryopreserved high cell density NK cell enriched PBMCs post thaw at various days in culture were used as effector cells to evaluate their functionality. Cultured PBMCs, as well as revived cultured PBMC’s post cryopreservation were centrifuged at 443 x g for 10 minutes and supernatant was discarded. The cells were resuspended in the assay media to each of the wells to attain a final density of 25 x 104 cells/well except for the media control wells and spontaneous lysis wells. The plate was incubated at 37 °C ± 1 °C and 5 ± 0.5% CO2 for 4.0 ± 0.5% h. 100 µl of CytoTox-ONETM reagent (Promega Corporation USA), sufficiently pre-incubated at 37 °C ± 1 °C and diluted with 11 mL of buffer, was added to each well and the plate was incubated for 25 min at 25 ± 2 °C with shaking at 300 rpm. The plate was removed and 50 µL of stop solution was added to all the wells and the fluorescence was measured at excitation wavelength 560 nm and emission wavelength 590 nm with cut off at 590 nm. The data obtained was plotted on a 4 parametric logistic (4-PL) graphs of concentration vs RFUs. The percent relative potency of the sample was calculated against the reference standard using restricted model and an average of three independent repeats was reported.
Results of the assay are measured in terms of relative potency and are represented in figure 1 and 2.
From the figure 2, it is evident that, cultured human PBMC’s (post freezing, thawing and subsequently cultured) as well as unfrozen freshly cultured PBMCs on different days in culture were produced comparable effector functions.
Example 5 Assessment of functionality of high cell density PBMCs as source effector cells using various afucosylated glycoforms in ADCC
Effect of various glycoforms containing antibody on ADCC assay was already established in the art. However, whether these overnight rested frozen PBMCs were sensitive or in other terms functional to the effect of these glycoforms on ADCC or not was evaluated using different afucosylation containing antibodies.
This experiment was performed as described in example 5. Results of the assay is measured in terms of % relative ADCC activity and is represented in figure 4.
Form the figure 4, it is evident as the amount of afucosylation content in antibody increases percentage of relative ADCC activity is also increased which indicates the overnight rested PBMCs are functional in ADCC assay.
,CLAIMS:We Claim:
1. A method of performing cryopreservation of high cell density peripheral blood mononuclear cells (PBMC) comprising,
a) adding a freezing mixture comprising about 4 to about 6% of hydroxy ethyl starch (HES), Di methyl sulfoxide (DMSO), pooled human serum and growth medium to cultured PBMC
b) cooling the said solution at 1ºC/min in a cooler and storing at -80 ºC.
2. A method according to claim 1 wherein, the concentration of HES in freezing mixture is about 4 to about 4.5%.
3. A method according to claim 1 wherein, the concentration of DMSO is about 10% (v/v).
4. A method according to claim 1 wherein, the said growth media comprises 10% Lymphocyte Growth Medium-3 (LGM3) with 50ng/ml Recombinant human interleukin-2 (rhuIL2).
5. A method according to claim 1 wherein, the concentration of said pooled human serum is about 50% (v/v).
6. A method according to claim 1 wherein, the said density of PBMC is in the about 107 cells to about 108 cells.
7. A method according to claim 1 wherein, biological activity of cryopreserved PBMCs upon thawing and subsequent culturing is comparable to freshly cultured PBMC when used in a functional assay such as ADCC.