Cell Freezing Kits and Their Use Thereof

Abstract

The disclose provides a cell freezing kit having a device equipped with a filter, and a container having hermetic closing means, which is capable of containing the device equipped with a filter, and methods of use thereof.

Claims

1-22. (canceled)

23. A cell freezing kit, comprising: (i) a device equipped with a filter; and (ii) a container having hermetic closing means, which is capable of containing the device equipped with a filter, wherein the filter has hydrophobic properties and/or the kit also comprises a hydrophobic solution.

24. The cell freezing kit of claim 23, wherein the average diameter of the pores of the filter is between 2 and 10 microns.

25. The cell freezing kit of claim 23, wherein the filter has hydrophobic properties so that the freezing medium does not pass through the filter in the absence of a driving force greater than gravitational force.

26. The cell freezing kit of claim 23, which comprises a hydrophobic solution capable of passing through the filter only when a driving force greater than gravitational force is applied thereto.

27. The cell freezing kit of claim 26, wherein the hydrophobic solution is a solution of glycerol.

28. The cell freezing kit of claim 26, wherein the hydrophobic solution is a sterile oil free of endotoxins.

29. The cell freezing kit of claim 23, wherein the device equipped with a filter is a centrifugal filter.

30. The cell freezing kit of claim 23, wherein the hermetic closing means of the container comprise a screw cap.

31. The cell freezing kit of claim 23, wherein the assembly of the container and the device equipped with a filter constitutes a centrifugation unit suitable for centrifugation between 50 and 1500 g.

32. The cell freezing kit of claim 31, which comprises one or several centrifugation units, wherein said centrifugation units are sterile or sterilizable.

33. A method for freezing cells using the device of claim 23, comprising: (i) introducing cells in a freezing medium in the device equipped with a filter; (ii) putting the device equipped with a filter into the container; (iii) hermetically closing the container; and (iv) placing the container at a freezing temperature.

34. The method of claim 33, wherein step (ii) is performed before step (i).

35. The method of claim 33, wherein a drop of a hydrophobic solution is added to the filter before step (i).

36. The method of claim 33, wherein the device equipped with a filter and the container are sterilized before step (i).

37. The method of claim 33, wherein the amount of cells placed in each device equipped with a filter is between 10.sup.4 and 10.sup.7.

38. A method for thawing cells frozen using the device of claim 23, comprising: (i) placing the container, containing a frozen cell suspension in a device equipped with a filter, at a temperature which allows the cell suspension to thaw; (ii) applying to the cell suspension a force which drives the passing of the freezing medium through the filter; (iii) resuspending the cells in cell medium.

39. The method of claim 38, wherein step (ii) is performed by centrifugation.

40. The method of claim 39, wherein the centrifugation is performed during less than or equal to 10 minutes, at a speed which makes it possible to apply to the cell suspension an acceleration of between 50 and 1500 g, preferably between 100 and 200 g.

Description

FIGURE LEGENDS

[0037] FIG. 1: Number of cells recovered after thawing and centrifugation in Ultrafree tubes.

[0038] FIG. 2: Yield and cell viability of cells thawed and centrifuged in Ultrafree tubes.

[0039] FIG. 3: Number of cells per well during the cell culture (cells thawed according to the Ultrafree tube technique and seeded in a 4-well plate).

[0040] FIG. 4: Cell viability during culture (cells thawed according to the Ultrafree tube technique and seeded in a 4-well plate).

[0041] FIG. 5: VERO cell viability after 10 days of freezing at 80 C. in Ultrafree-CL tubes, the membranes of which have been covered with 50 l of glycerol.

[0042] FIG. 6: Cell amplification after freezing in Ultrafree-CL tubes.

EXAMPLES

Example 1: Use of the Thawing Columns (=Centrifugal Filters) for Thawing and Culturing Cryopreserved Cells

Description of the Centrifugal Filters

[0043] Ultrafree-CL SV centrifugal filters from Millipore: [0044] reference: UFC40SV25 Ultrafree-CL SV 5.0 centrifugal filters [0045] filter for microfiltration [0046] hydrophilic PVDF (polyvinylidene fluoride) filter [0047] filter pore size: 5.0 m [0048] polypropylene device [0049] non-sterile.

Prior Process on the Centrifugal Filters

[0050] In order to make them usable in cell culture, these centrifugal filters underwent the following treatment: [0051] welded individual polyethylene packaging (packaging can optionally be in several units) [0052] sterilization by irradiation with gamma-rays at 25 kGy (an amount of 8 kGy is sufficient).
The embryo toxicity of this device thus treated was tested by means of Mouse embryo assays, demonstrating the innocuousness of the centrifugal filters on mouse embryo development up to the blastocyst stage.
Cells: monkey kidney cell line: Vero cells, cryopreserved in nitrogen vapor, at 210.sup.6 cells/vial, with 1 ml of freezing medium: MEM culture medium (80%)/fetal calf serum (10%)/dimethyl sulfoxide (10%).

Protocol:

Thawing of the Cryopreserved Cells

[0053] Using one vial of cryopreserved VERO cells (Vero WHO, catalogue No. 88020401 at the ECACC) [0054] Place the vial in a water bath at 37 C. for 1 minute [0055] Homogenize the content of the vial [0056] Deposit 500 l of cell suspension in each of the two sterilized UltraFree tubes [0057] Add 1 ml of MEM medium to each of the centrifugal filters [0058] Centrifuge the Ultrafree centrifugal filters for 5 min at 100 g [0059] During the centrifugation: [0060] Prepare 1 15 ml tube (tube T1) [0061] Prepare 1 hemolytic tube containing 100 l of trypan blue (tube T2).
Recovery of the cells in the Ultrafree tubes at the end of centrifugation: [0062] Deposit 500 l of CCM-30 medium (Vitrolife, Goteborg, Sweden) on the filter of the first Ultrafree tube [0063] Carry out a series of 5 suction/discharge cycles on the whole of the filer [0064] Deposit the 500 l of obtained suspension in the tube T1 [0065] Deposit a further 500 l of CCM medium on the filter of the same first Ultrafree tube [0066] Carry out a series of 5 suction/discharge cycles on the whole of the filter [0067] Deposit the 500 l of obtained suspension in T1 [0068] Carry out exactly the same manipulation on the second centrifugal filter. [0069] Homogenize the suspension of T1: Suspension S1 (volume 2 ml) [0070] Sample and deposit 100 l of the cell suspension S1 in T2 for counting [0071] Perform the cell count, and viability, by depositing the cell suspension between slide and cover slip of a hemocytometer [0072] Adjust the cell concentration to 480 000 cells/ml with CCM medium [0073] Seed 4 wells of an IVF plate at 240 000 cells/well (i.e. 500 l/well).

[0074] Monitoring of the culture on D1 [0075] Observe the cultures [0076] Trypsinize one well and perform a count/viability (+trypan blue) [0077] Rinse the wells with 1 ml of PBS [0078] Suction of the PBS [0079] Add 300 l of trypsin and place the cultures at 37 C. for 5 minutes [0080] Suction and discharge (using a P200 and 200 l tips) of the suspension until complete dissolution of the cell clumps [0081] Inhibit the action of the trypsin by adding 200 l of medium for VERO cells [0082] Suction and discharge of the suspension until complete dissolution of the cell clumps [0083] Count the VERO cells using a hemocytometer. [0084] Rinse the other wells with 1 ml of PBS/well. [0085] Add 1 ml of CCM/well [0086] Incubate the plates at 37 C.+5% CO.sub.2.
Monitoring of the culture on D2=observation of the cultures and trypsinization of one well according to the description idem D1 (no change of medium). [0087] Monitoring of the culture on D5=observation of the cultures and trypsinization of one well according to the description idem D1 (no change of medium). [0088] Monitoring of the culture on D7=observation of the cultures and trypsinization of one well according to the description idem D1 (no change of medium).

Results

[0089] The experiment was carried out on three vials of VERO cells, and the results obtained are summarized in the graphs of FIGS. 1 and 2.

[0090] The results obtained show that the system for thawing VERO cells using the centrifugal filters makes it possible to recover more than one million live cells per vial of cells thawed. The cell viability is very satisfactory (greater than 90%), and the yield is also satisfactory (greater than 50%).

[0091] The results from placing back in culture the VERO cells thawed according to this Ultrafree centrifugal filter technique show a very good cell viability over time of greater than 90% over the course of 7 days of culture (FIGS. 3 and 4).

[0092] Moreover, the number of cells per well over time shows good cell growth starting from day 1. The drop in the number of cells between D0 and D1 after thawing and placing back in culture is observed in many cell types; it is explained by the fact that some cells may be viable at thawing without being capable of subsequently proliferating.

[0093] Starting from day D1, the number of cells per well gradually increases, and finally reaches close to 0.5 million per well on day 7 of culture, i.e. double the number of cells seeded.

[0094] The Vero cells thawed according to the Ultrafree tube technique are therefore viable and capable of proliferating in culture for 7 days. The results obtained on three tests show that this technique is reliable and reproducible.

Example 2: Use of the Thawing Columns (Centrifugal Filters) in a Ready-to-Use Kit for Thawing and Preparing VERO Cells for Mouse Embryo Development up to the Blastocyst Stage

Protocol:

[0095] On day D0: thaw one vial of VERO cells and seed 4 wells of an IVF plate, according to the protocol described in example No. 1. [0096] On day D1: rinse each well with 1 ml of a buffer solution, and add 1 ml of CCM medium. [0097] Deposit mouse embryos at the zygote stage: 10 embryos/well. [0098] On day D2, evaluate the % of zygotes having reached the two-cell embryo stage. [0099] On day 5: evaluate the % of 2-cell embryos having reached the blastocyst stage. [0100] On day 6: evaluate the % of 2-cell embryos having reached the blastocyst stage.

Results

[0101] The experiment was carried out on three vials of VERO cells, and the results obtained are summarized below.

TABLE-US-00001 TABLE 1 Day 2 Day 5 2-cell embryo / Blastocyst / 2-cell zygote embryo Vial 1 97% 96% Vial 2 97% 96% Vial 3 97% 97% Average 97% 96%
The results obtained show that the system of thawing VERO cells using the centrifugal filters makes it possible to produce a cell layer suitable for the development of mouse embryos up to the blastocyst stage, and said embryos are also capable of growing. The blastulation rate on the cell layer of Vero cells is very satisfactory: more than 90%.

Example 3: Freezing/Thawing the Cells in One and the Same Container: Ultrafree Centrifugal Tube

[0102] Freshly trypsinized Vero cells are frozen directly in the filter column of the Ultrafree tube (often incorrectly called the filter). The cells are then thawed in these same tubes.

Materials:

[0103] Description of the centrifugal filters: cf. example 1. [0104] Prior process on the centrifugal filters: cf. example 1. [0105] The glycerol used is the one from Euromedex, ref. 50405, purity >99.5%, molecular biology and electrophoresis grade. Cell culture glycerol can of course be used in place of the one used here, as can any liquid that is water-immiscible and compatible with cell survival/cell culture. [0106] Cells: monkey kidney cell line: Vero cells, cryopreserved in nitrogen vapor, at 210.sup.6 cells/vial, with 1 ml of freezing medium: MEM culture medium (80%)/fetal calf serum (10%)/dimethyl sulfoxide (10%). [0107] Culturing of cells: MEM culture medium (95%)/fetal calf serum (5%).

Protocol:

Freezing of the Cell Suspension in the Ultrafree Tube Containing the Centrifugal Filter

[0108] Starting from a suspension of freshly trypsinized cells in culture (viability measured=viability before freezing): [0109] Prepare a suspension at 0.5 million/ml in freezing medium composed of 70% MEM- (Gibco), 20% FCS, 10% DMSO; [0110] Deposit 50 l of glycerol on the centrifugal filter; [0111] Deposit thereon 1 ml of the cell suspension in the freezing medium; [0112] Prepare 3 centrifugal tubes in this way; [0113] Close the Ultrafree tubes and place them at 80 C.

Thawing of the Cryopreserved Cells in the Same Ultrafree Tube Containing the Centrifugal Filter

[0114] Place the 3 Ultrafree tubes containing the cell suspension in a water bath at 37 C. for 1 minute; [0115] Centrifuge the tubes for 5 min at 100 g.

Recovery of the Cells on the Filter of the Ultrafree Tubes at the End of Centrifugation:

[0116] Deposit 500 l of 95% MEM- (Gibco), 5% FCS culture medium on the Ultrafree tube filter; [0117] Carry out a series of 5 suction/discharge cycles on the whole of the filter: [0118] Deposit a further 500 l of culture medium on the Ultrafree tube filter; [0119] Carry out a series of 5 suction/discharge cycles on the whole of the filter; [0120] Place 1 ml final of the cell suspension in a 15 ml tube; [0121] Sample and deposit 100 l of the cell suspension for counting; [0122] Perform the cell count, and viability, by depositing the cell suspension between slide and cover slip of a hemocytometer; [0123] Seed in a 75 cm.sup.2 flask and incubate the flasks at 37 C.+5% CO.sub.2.

[0124] Result:

[0125] The entire test is reproduced three times, and the viability results are reported in FIG. 5. A viability of about 88.0% (4.9) was obtained. FIG. 6 confirms that this technique is reliable and reproducible, the cells showing a good proliferation capacity after thawing.