System for storing and transporting cell lines

Abstract

The present invention relates to a system for in vitro storage and transport of cell lines based on a combination of a specific cell transport medium and a gelatin matrix. The system of the present invention is particularly suitable for transporting HEK-293 and Caco-2 cell lines, as their viability remains substantially unchanged during storage and / or transport for at least 96 hours. The present invention also relates to a method for in vitro transport of cell lines, as well as a kit for transporting said cell lines.

Description

[Technical Field]

[0001] The present invention relates to the field of cell line storage and transport. More specifically, the present invention relates to a system for in vitro storage and transport of cell lines based on a combination of a specific cell transport medium and a gelatin matrix. The system of the present invention is particularly suitable for transporting HEK-293 and Caco-2 cell lines, as their viability and viability remain substantially unchanged during storage and / or transport for at least 96 hours. The present invention also relates to a method for in vitro transport of cell lines and a kit for transporting said cell lines. [Background technology]

[0002] Cell cultures, whether homogeneous or not (co-cultures), can constitute useful models of several genetic, biochemical, metabolic or physiological processes occurring in living organisms. Their ease of use allows a large number of conditions to be analyzed before carrying out definitive experiments on animals or clinical assays on humans. In vitro models constitute tools for validating new therapeutic targets, for selecting seeds in sophisticated systems, for defining the mechanism of action of new molecules, and generally for biomedical, biotechnological or cosmetic research.

[0003] Generally, all cell culture-based models have a limited shelf life. Thus, cultured cells undergo various differentiation stages and require continuous manipulation to maintain the properties that make them suitable models. These limitations in the manipulation and generation of different in vitro cell models make them impractical for occasional users and generally limit the commercialization of the models in their final form.

[0004] Additionally, when cell lines are transported, cell viability decreases over time, making the transport of certain cell lines unfeasible for commercial sale in a ready-to-use format. Thus, there is a need to develop cell line transport systems and / or cell line transport media that improve cell survival time and cell viability to allow for ease of storage and transport, and thus enable the commercialization of these ready-to-use cell models.

[0005] Some prior art documents, such as European Patent No. 1650292, describe solutions to improve the transport of cell lines. However, the transport systems and methods described in this prior art document are not optimal for transporting all types of cell lines. For example, the HEK-293 cell line is very sensitive and has a viability of more than 24 hours, which makes it impossible to market it in a ready-to-use format.

[0006] Therefore, there is a need to develop new cell line delivery systems that can extend the survival and viability of the most sensitive cell lines, such as HEK-293, for more than 48 hours, preferably more than 72 hours, so that this type of cell can be commercially available in a ready-to-use format.

[0007] The present invention provides an in vitro storage and transport system that allows the survival and viability of various cell lines, in particular the HEK-293 cell line, for periods of more than 72 hours, and even up to 96 hours, and in principle allows the transport of these types of cells in a ready-to-use format to any continent. The system of the present invention has proven particularly suitable and useful for transporting HEK-293 and Caco-2 cell lines. Summary of the Invention

[0008] The main object of the present invention is to provide a system for in vitro storage and transport of cell lines, comprising: a) i.Leibovitz L-15 basal medium ii.FBS iii.HEPES iv. Glutamine v. A mixture of penicillin and streptomycin and a cell transport medium containing b) a solidified gelatin matrix and It is a system including

[0009] Hereinafter, this storage and / or transport system will be referred to as the system of the present invention.

[0010] Another object of the present invention is a method for the in vitro storage and / or transport of cell lines based on the use of the system of the present invention, which method will be referred to as the method of the present invention.

[0011] A further object relates to a kit comprising the system of the invention and a support for immobilizing the cell line to be transported, which will be referred to as the kit of the invention.

[0012] A final object of the invention is represented by the use of the system according to the invention for storing and / or transporting cell lines, preferably for transporting the HEK-293 and Caco-2 cell lines. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 compares the cell viability of mock HEK-293 cells after exposure to the delivery system of the present invention and to the normal medium (D6046) for culturing these cells. [Figure 2] FIG. 2 compares HEK-293 MATE1 cell viability after exposure to the delivery system of the present invention and to the normal medium (D6046) for culturing these cells. [Figure 3] FIG. 3 is a comparison of the maintenance of cell monolayer integrity in Caco-2 cells after exposure to the delivery system of the present invention compared to the current Caco-2 cell delivery system (CacoReady). DETAILED DESCRIPTION OF THE INVENTION

[0014] A system for in vitro storage and transport of cell lines. A main aspect of the present invention is a system for in vitro storage and transport of cell lines, comprising: a) i.Leibovitz L-15 basal medium ii.FBS iii.HEPES iv. Glutamine v. A mixture of penicillin and streptomycin and a cell transport medium containing b) a solidified gelatin matrix and This refers to a system that includes:

[0015] In a more specific and preferred manner, the system for in vitro storage and transport of cell lines of the present invention comprises: a) i. 78% to 88.5% by volume of Leibovitz L-15 basal medium ii. 8% to 12% by volume of FBS iii. 2.5% to 6% by volume of HEPES iv. 0.5% to 2% glutamine by volume v. 0.5% to 2% by volume of a mixture of penicillin and streptomycin a cell transport medium comprising: b) a solidification matrix of 10-50 mg of gelatin per ml of transport medium; Includes:

[0016] The storage and transport system of the present invention surprisingly demonstrated the ability to maintain cell viability for more than 72 hours. In the case of HEK-293 cells, which previously could not support transport times longer than 24 hours, at least 80% cell viability was observed after 96 hours. In the case of Caco-2 cells, the storage and transport system of the present invention allowed for transport of the cells at room temperature and was observed to maintain monolayer integrity unchanged for up to at least 11 days.

[0017] pH is one of the major parameters affecting the viability of cell lines, especially HEK-293 and Caco-2 cells. The present authors observed that the combined action of the buffering agent HEPES and the Leibovitz L-15 basal medium provides the system of the present invention with the ability to maintain a stable physiological pH such that cell survival and viability are significantly improved.

[0018] Another essential element of the storage and transport system of the present invention is the solidified gelatin matrix, which allows for the protection of the cells and the maintenance of their functional properties. Gelatin also ensures proper gas exchange of the cells.

[0019] The gelatin used is preferably Type A gelatin, although any type of natural or commercially available gelatin can be used in connection with the system of the present invention.

[0020] As mentioned above, the system of the present invention allows for the transport of any cell type, but is particularly suitable for storing and transporting cell lines that are particularly sensitive to stress situations. In this regard, the system is adapted and particularly suitable for transporting HEK-293 cells as well as Caco-2 cells.

[0021] To use the system of the present invention, cells must first be immobilized on a support for transport. Any type of support that allows cell transport can be used. In certain embodiments, the support is selected from Petri dishes, multiwell plates, transwell membrane inserts, biomimetic systems, synthetic supports for 3D culture, glass plates, slides, or any type of support that has been previously coated with, for example, collagen, polylysine, or other similar elements to promote cell adhesion.

[0022] When the cells reach their appropriate functional state and appropriate confluence, they are ready to be coated with the system of the present invention. In the context of the present invention, appropriate functional state is understood to be the state of the living cells of the culture in which they can perform the functions assigned to them for the assay of interest. Once they reach said appropriate functional state and appropriate confluence, the system of the present invention is added, which, thanks to the gelatin, ensures that the cells are completely immobilized and maintain their functional state until they reach their final destination. Gelatin limits and reduces the mechanical stress experienced by the cells during the transport process.

[0023] The combined effect of the transport medium reducing physiological stress as well as gelatin reducing mechanical stress on the cells results in a synergistic effect that provides the appropriate transport conditions for cells to reach their destination in their optimal functional state.

[0024] The system of the present invention has the advantage that it does not require special shipping conditions and in fact allows shipping of cell lines at room temperature, which represents an advantage over other systems that require refrigeration or special shipping conditions.

[0025] Methods for storing and / or transporting cell lines A second aspect of the present invention provides a method for in vitro storage and / or transport of a cell line, comprising the steps of: A) Cell lines immobilized on a support are a) i.Leibovitz L-15 basal medium ii.FBS iii.HEPES iv. Glutamine v. A mixture of penicillin and streptomycin and a cell transport medium containing b) a gelatin matrix coating with a system comprising B) solidifying the gelatin matrix at a temperature between 15°C and 25°C; C) storing and / or transporting the cell lines at a temperature between 15°C and 25°C for up to 96 hours; The present invention relates to a method, including:

[0026] In certain embodiments, the method comprises: a) i. 78% to 88.5% by volume of Leibovitz L-15 basal medium ii. 8% to 12% by volume of FBS iii. 2.5% to 6% by volume of HEPES iv. 0.5% to 2% glutamine by volume v. 0.5% to 2% by volume of a mixture of penicillin and streptomycin and a cell transport medium containing b) a matrix of 10–50 mg of gelatin per ml of transport medium; This includes using

[0027] The first step (Step A) of the method involves preparing and housing the cell culture to be transferred. To this end, cells are seeded at a density determined for each cell type before coating them with the system of the present invention. Depending on the type of assay they are intended for, cell lines are seeded on one type of support or another. The cultures must be maintained as long as necessary to reach proper function and confluence, preferably with medium changes every 48-72 hours as needed.

[0028] Any suitable support for cell transport can be used, but in particular the supports used are chosen from Petri dishes, multiwell plates, transwell membrane inserts, biomimetic systems, synthetic supports for 3D culture, glass plates, slides or any type of support pre-coated to promote cell adhesion, for example by coating with collagen, polylysine or other similar elements.

[0029] Once the cell line has reached a suitable functional state, it is coated with a system according to the invention, comprising a transport medium and a gelatin solution, which must be in a liquid state, according to step A of the method. The gelatin solution is prepared by dissolving gelatin in the transport medium, which acts as a solvent. By using the transport medium as a solvent, it is achieved that the cell line stored and / or transported according to the method of the invention maintains the functional properties of the culture, so that it is ready for use once it reaches its final destination.

[0030] The gelatin used is preferably Type A gelatin, although any type of natural or commercially available gelatin can be used in connection with the system of the present invention.

[0031] In step B), the gelatin matrix is ​​solidified at a temperature between 15° C. and 25° C., thereby immobilizing and stabilizing the cells. The gelatin layer represents a physical protection for the cells, allowing them to better withstand the mechanical stresses arising from the transport process.

[0032] The transportation (stage C) is carried out at a temperature between 15°C and 25°C, allowing transport for more than 48 hours, preferably more than 72 hours, preferably up to 96 hours.

[0033] Once the cultures reach their destination and are to be used, the plates are incubated with the solid gelatin in a cell incubator for 1.5-4 hours at 37°C, 90% humidity, and 5% CO2 until completely liquefied. It is then removed by aspiration, and culture medium specific to the cells in question is applied. The cells are then incubated at 37°C, 90% humidity, and 5% CO2 until use.

[0034] The method of the present invention is applicable to any type of cell line, but is particularly suitable for storing and / or transporting cell lines selected from HEK-293 and Caco-2.

[0035] Cell Line Transport Kit In another aspect, the present invention provides a kit for in vitro transport and storage of a cell line, comprising: a. A system for in vitro storage and / or transport of cell lines according to the invention; b. A support for immobilizing the cell line to be transported The present invention relates to a kit comprising:

[0036] The cell storage and transport system of the kit of the present invention includes both a transport medium and a gelatin matrix under the above terms and parameters.

[0037] With regard to the support, the kit may comprise any suitable support for cell transport, but in a preferred embodiment the support used is selected from Petri dishes, multiwell plates, transwell membrane inserts, biomimetic systems, synthetic supports for 3D culture, glass plates, slides or any type of support pre-coated with, for example, a coating with collagen, polylysine or other similar elements to promote cell adhesion.

[0038] The kit of the present invention is in principle useful for the transport of any type of cell line, but is particularly suitable for storing and / or transporting cell lines selected from the HEK-293 cell line and the Caco-2 cell line.

[0039] use A final aspect of the present invention is represented by the use of the system of the present invention.

[0040] The system of the present invention allows in principle the storage and / or transport of any cell line, but has been specially designed and developed for the transport of cell lines that are most sensitive to stress situations.

[0041] Preferably, the system of the present invention is useful for storing and / or transporting HEK-293 and Caco-2 cell lines.

[0042] The system of the present invention has been observed to maintain 100% cell viability of cells for 72 hours, and to enable cells to maintain at least 80% cell viability after 96 hours or more depending on the cell type. Thus, the system of the present invention practically enables the shipping and transport of cells, and particularly sensitive cells such as HEK-293 and Caco-2, to any location on any continent.

[0043] The following examples serve to illustrate the present invention but are not intended to limit the scope of the invention in any way. [Example]

[0044] Example 1: Cell viability of mock HEK-293 and HEK-293 MATE1 cells A series of assays were performed on mock HEK-293 and HEK-293 MATE1 cells to test cell viability after 72 hours (3 days) and 96 hours (4 days) using different delivery methods. As can be seen in this comparative study, the improved survival effect of the delivery composition of the present invention was demonstrated over the maintenance medium (initial delivery medium) commonly used for this type of cell.

[0045] The following formulations were assayed: Initial transport medium without Hepes (D6046+0% H): -DMEM D6046 low glucose 85.5% (Sigma); -10% FBS(Biowest); -L-glutamine 1% (Lonza); Pen / Strep 1% (Lonza); and -Type A gelatin 25mg / ml (Sigma).

[0046] Initial transport medium + 5% Hepes (D6046 + 5% H): -DMEM D6046 low glucose 80.5% (Sigma); -10% FBS(Biowest); -Hepes 5% (Gibco); -L-glutamine 1% (Lonza); Pen / Strep 1% (Lonza); and -Type A gelatin 25mg / ml (Sigma).

[0047] Fresh transport medium without Hepes (L15 + 0% H): -Leibovitz L-15 medium 85.5% (Sigma); -10% FBS(Biowest); -Glutamine 1% (Lonza); -Penicillin-streptomycin 1% (Lonza); and -Type A gelatin 25mg / ml (Sigma).

[0048] Fresh transport medium + 5% Hepes (L15 + 5% H): -Leibovitz L-15 medium 80.5% (Sigma); -10% FBS(Biowest); -Hepes 5% (Gibco); -Glutamine 1% (Lonza); -Penicillin-streptomycin 1% (Lonza); and -Type A gelatin 25mg / ml (Sigma). Note: Volume / Volume %

[0049] A comparative study was performed on cells. Both mock HEK-293 cells and HEK-293 MATE1 cells were seeded at a density of 50,000 cells / well on poly-D-lysine-coated 96-well plates. 24 hours after seeding, when full confluence was reached, the medium was removed and 200 μl / well of transport medium was added. The plates were allowed to settle in a flow chamber, sealed with parafilm, and then placed in a climate chamber at 23 °C.

[0050] Plates were liquefied after 72 and 96 hours of exposure to transport medium. The transport medium was liquefied by placing the plates in a CO2 incubator at 37°C for 90 minutes. Once the gelatin was liquefied, the transport medium was removed and HEK-293 maintenance medium was applied (100 μl / well).

[0051] After 24 hours, cell viability was assayed using Alamar Blue (110 μl / well of 10% Alamar Blue in HEK-293 maintenance medium, 2 hours incubation in a CO 2 incubator).

[0052] The percentage of cell viability was calculated relative to the net value of RFU (relative fluorescence units) of control cells at time 0 before application of transport medium (TM): Survival % = 100*(Net RFU after TM) / (Net RFU before TM)

[0053] Significant differences between groups were verified using Student's t-test (unpaired). A value of p≦0.05 was considered statistically significant. Tables 1 and 2 below show the results of comparative assays for both mock HEK-293 cells and HEK-293 MATE1 cells. The results can also be observed in Figure 1 (Mock HEK-293) and Figure 2 (HEK-293 MATE1). TIFF0007763177000001.tif101170

[0054] Results using mock HEK-293 cells already demonstrate that, in the absence of HEPES buffer, the use of Leibovitz L15 basal medium versus DMEM D6046 provides a clear improvement in survival at 72 h. This difference is minimized when HEPES is added to DMEM D6046 medium but not to Leibovitz L15 basal medium. However, when HEPES is added to L15 medium, the difference from DMEM D6046 medium is highly significant. Significant differences are also observed when comparing L15 medium with or without HEPES, demonstrating the importance of this buffer for cell survival. However, taken as a whole, these results suggest that the combination of Leibovitz L-15 basal medium and HEPES buffer is crucial for the survival of mock HEK-293 cells after 72 h.

[0055] At 96 hours, the results were similar, but the differences were highlighted, with the use of Leibovitz L-15 basal medium containing 5% HEPES resulting in a highly significant increase in cell viability compared to the other controls. TIFF0007763177000002.tif103170

[0056] Results in HEK-293 MATE1 cells after 72 hours are similar to those in mock HEK-293 cells, but the effect of HEPES on survival is observed to be more significant in all comparisons.

[0057] At 96 hours, a similar pattern is observed, but it can be inferred that the relative importance of L-15 basal medium to the survival of HEK-293 MATE1 cells is more important in our system than in mock HEK-293 cells. This does not imply that HEPES is unimportant for survival after 96 hours, but again suggests a synergistic effect of using HEPES buffer together with Leibovitz L-15 basal medium in significantly improving cell viability at both 72 and 96 hours in HEK-293 MATE1 and mock HEK-293 cells.

[0058] In conclusion, the L-15 + 5% HEPES compositions assayed allow cell transport at room temperature and maintain cell viability substantially unchanged for up to 4 days.

[0059] After 4 days of exposure to room temperature, transport medium based on L15 + 5% HEPES maintains 100% and 80% cell viability of HEK-293 Mock and MATE1, respectively.

[0060] Example 2: Assessment of cell integrity in Caco-2 cells A series of assays were performed to assess the integrity of Caco2 cell monolayers over 4 days (96 hours) and 11 days (264 hours) using different delivery methods. Currently available delivery methods allow for a maximum of 4 days of travel, so it was desirable to test whether the delivery system of the present invention could overcome this time barrier. As seen in this comparative study, the delivery composition of the present invention demonstrated improved survival benefits over current methods. To this end, cell monolayer integrity was assessed after the test period by measuring the transepithelial resistance (TEER) of the cell monolayer.

[0061] The following formulations were assayed: Current Transport Composition (SM CR): ·DMEM low glucose 85.5% (Sigma); · 10% FBS (Biowest); L-glutamine 1% (Lonza); and · Penicillin-streptomycin 1% (Lonza); · Type A gelatin 25mg / ml (Sigma).

[0062] Transport composition of the present invention (SM L15): · L-15 medium (Leibovitz) containing glutamine 80.5% (Sigma); ·FBS 10% (Biowest); ·Hepes 5% (Gibco); · L-glutamine 1% (Lonza); Penicillin-streptomycin 1% (Lonza); and · Type A gelatin 25mg / ml (Sigma).

[0063] Caco Maintenance Medium (used for seeding and maintaining plates): DMEM low glucose (D6046 Sigma) FBS 10% (Biowest) L-Glutamine 1% (Lonza) Penicillin-streptomycin 1% (Sigma) Note: Volume / Volume %

[0064] The following equipment was used to perform the tests: -Biological cabinet -CO2 incubator -Climate chamber -Epithelial Voltage / Ohm (TEER) Meter - EVOM2 -STX100C96 specific electrode for measuring transepithelial electrical resistance in Corning brand 96-well Transwell plates

[0065] A comparative study was performed on Caco2 cells according to the internally defined manufacturing procedure for the CacoReady 96-well product.

[0066] Cells were seeded into four 96-well plates (#3392, Corning) at a density of 11,000 cells / well. Cultures were maintained for 13 days, with medium changes approximately every 48 hours.

[0067] On day 14 after seeding, after reaching full confluence, the medium was removed and 75 μL of transport medium was added to the apical compartment and 235 μL to the basal compartment. The plates were warmed in a flow cabinet, sealed with parafilm, and then placed in a climate chamber at 23°C.

[0068] Plates were liquefied after 4 days of exposure to transport medium (day 18) and after 11 days of exposure (day 25). The transport medium was liquefied by placing the plates in a CO2 incubator at 37°C for 4 hours. Once the transport medium was liquefied, the transport medium was removed and Caco2 maintenance medium was applied (75 μL / apical + 235 μL / basal).

[0069] 48 hours after liquefaction, medium changes are performed every 2 days until days 25 and 32 during which the transepithelial resistance (TEER) of the cell monolayer is measured randomly to check the integrity of the cell barrier. Table 3 summarizes this schedule. TIFF0007763177000003.tif77170

[0070] Transepithelial resistance is calculated by multiplying the net TEER value by the membrane area of ​​the seeded well. TEER=Ω*cm2 Corning 96-well plate reference #3392 has a membrane area of ​​0.143 cm 2 is equal to CacoReady product specifications: TEER ≥ 500Ω.cm 2 Establish the correct state of the cell monolayer for measurements.

[0071] The results obtained for the transepithelial resistance of cell monolayers for the current transport medium (SM CR) and the transport system of the present invention (SM L15) are detailed in Table 4 for periods of 4 and 11 days, respectively. These results are also detailed graphically in Figure 3. TIFF0007763177000004.tif34170

[0072] As can be seen, the current vehicle (SM CR) is unable to maintain the integrity of the monolayer for 11 days as the TEER results are outside the established limit specifications.

[0073] In contrast, these results demonstrate that the system of the present invention (SM L15) allows the transport of Caco2 cells at room temperature and maintains the monolayer integrity unchanged for at least 11 days, which represents a significant improvement compared to currently existing compositions.

Claims

1. A composition for in vitro storage and transport of cell lines, comprising: a) i. 78% to 88.5% by volume of Leibovitz L-15 basal medium ii. 8% to 12% by volume of FBS iii. 2.5% to 6% by volume of HEPES iv. 0.5% to 2% by volume of glutamine v. 0.5% to 2% by volume of a mixture of penicillin and streptomycin and a cell transport medium containing b) a solidified matrix of 10-50 mg of gelatin per ml of transport medium; A composition comprising:

2. 2. The composition of claim 1, wherein the cell line is selected from the group consisting of HEK-293 and Caco-2 cell lines.

3. 3. The composition of claim 1, wherein the solidified matrix is ​​made of type A gelatin.

4. The composition of any one of claims 1 to 3, wherein the cell line is immobilized on a support for transport.

5. 5. The composition of claim 4, wherein the support is selected from a Petri dish, a multiwell plate, a membrane insert of a transwell, a biomimetic system, a synthetic support for 3D culture, a glass plate, a slide or any type of support pre-coated to promote cell adhesion.

6. 6. The composition of claim 1, wherein the delivery is at room temperature.

7. 1. A method for in vitro storage and / or transport of cell lines, comprising: A) A cell line immobilized on a support is a) i. Leibovitz L-15 basal medium ii. FBS iii. HEPES iv. glutamine v. A mixture of penicillin and streptomycin and a cell transport medium containing b) a gelatin matrix; coating with a composition comprising B) solidifying the gelatin matrix at a temperature between 15°C and 25°C; C) storing and / or transporting the cell lines at a temperature between 15°C and 25°C for up to 96 hours; A method comprising:

8. The composition, a) i. 78% to 88.5% by volume of Leibovitz L-15 basal medium ii. 8% to 12% by volume of FBS iii. 2.5% to 6% by volume of HEPES iv. 0.5% to 2% by volume of glutamine v. 0.5% to 2% by volume of a mixture of penicillin and streptomycin and a cell transport medium containing b) a matrix of 10-50 mg of gelatin per ml of transport medium; The method of claim 7, comprising:

9. 9. The method according to claim 7 or 8, wherein the support is selected from a Petri dish, a multiwell plate, a transwell membrane insert, a biomimetic system, a synthetic support for 3D culture, a glass plate, a slide or any type of support pre-coated to promote cell adhesion.

10. 10. The method of any one of claims 7 to 9, wherein the solidified matrix is ​​made of type A gelatin.

11. The method according to any one of claims 7 to 10, wherein the cell line to be stored and / or transported is selected from the group consisting of HEK-293 cell line and Caco-2 cell line.

12. A kit for in vitro transport and storage of cell lines, comprising: a. The composition according to any one of claims 1 to 3 and 5 to 6. b. Support for immobilizing cell lines Includes a kit.

13. 13. The kit of claim 12, wherein the support is selected from a Petri dish, a multiwell plate, a transwell membrane insert, a biomimetic system, a synthetic support for 3D culture, a glass plate, a slide, or any type of support pre-coated to promote cell adhesion.

14. 10. Use of a composition according to any one of claims 1 to 6 for the storage and / or transport of cell lines.

15. The use described in claim 14, wherein the cell lines are HEK-293 cell line and Caco-2 cell line.

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