Patent Description:
Cell cultures, homogeneous or not (co-cultures), can constitute useful models of some genetic, biochemical, metabolic or physiological processes that take place in a living organism. Their ease of use makes it possible to analyze a large number of conditions before carrying out the definitive experiments on animals or clinical assays on humans. In vitro models constitute a tool for validating new therapeutic targets, for selecting seeds in high-performance systems, for defining the action mechanism of new molecules and, in general, for biomedical, biotechnological or cosmetic research.

In general, all cell culture-based models have a limited shelf life. Thus, cultured cells go through various differentiation stages and require continuous manipulation to maintain the properties that make them a suitable model. These limitations in the manipulation and generation of the different in vitro cell models make them difficult to implement for occasional users and, in general, limit the commercialization of the models in their end format.

It must be added to this that, when transporting cell lines, cell survival decreases as the hours pass, which renders the transport of certain cell lines unviable for the commercial marketing thereof in a ready-to-use format. Therefore, there is a need to develop cell line transport systems and/or cell line transport media that make it possible to improve cell survival times and rates to facilitate the storage and transport and, therefore, the commercialization of these ready-to-use cell models.

Some prior art documents describe solutions for improving the transport of cell lines, such as for example <CIT>. However, the transport system and methods described in this prior art document are not optimal for transporting all types of cell lines. For example, the HEK-<NUM> cell line is highly sensitive and has a viability of more than <NUM> hours, which makes its commercial marketing in ready-to-use format impossible.

Some other solutions require special hypothermic conditions for the transport of cells such as the solutions provided in <NPL>and <CIT>.

Therefore, there is a need to develop new cell line transport systems that make it possible to extend the survival and viability rates of the most sensitive cell lines, such as HEK-<NUM>, beyond <NUM> hours and preferably beyond <NUM> hours so that this type of cells can be marketed in a ready-to-use format.

The present invention provides an in vitro storage and transport system that enables the survival and viability of different cell lines, particularly the HEK-<NUM> cell line, for periods greater than <NUM> hours and even up to <NUM> hours that, in principle, enables the transport of these types of cells in ready-to-use format to any continent. The system of the present invention has proven especially suitable and useful for transporting HEK-<NUM> cells and Caco-<NUM> cell lines.

The main object of the invention is a system for the in vitro storage and transport of cell lines comprising:.

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

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 invention. This method will be referred to as the method of the invention.

A further object refers to a kit comprising the system of the invention and a support for immobilizing the cell line to be transported. This will be referred to as the kit of the invention.

A last object of the invention is represented by the use of the system of the invention for storing and/or transporting cell lines and, preferably, for transporting HEK-<NUM> and Caco-<NUM> cell lines.

The main aspect of the invention refers to a system for the in vitro storage and transport of cell lines comprising:.

The storage and transport system of the present invention has surprisingly shown the ability to maintain cell viability beyond <NUM> hours. In the case of HEK-<NUM> cells, which to date were unable to support shipping times of more than <NUM> hours, cell survival rates of at least <NUM> % after <NUM> hours were observed. In the case of Caco-<NUM> cells, it has been observed that the storage and transport system of the present invention allows the transport of cells at room temperature, maintaining the integrity of the monolayer unchanged for up to at least <NUM> days.

pH is one of the main parameters that affects cell line viability and, in particular, HEK-<NUM> and Caco-2cells. The authors of the invention have observed that the joint action of HEPES, which is a buffer agent, and the action of the Leibovitz L-<NUM> basal medium provide the system of the invention with the ability to maintain a stable physiological pH so that the cell survival and viability are remarkably improved.

Another essential element of the storage and transport system of the invention is the solidified gelatin matrix. This allows protecting the cells, as a well as preserving their functional properties. Gelatin also guarantees a proper gas exchange of the cells.

Any type of natural or commercial gelatin can be used in the context of the system of the invention, although the gelatin used is preferably type A gelatin.

As mentioned above, the system of the invention allows any cell type to be transported, but is especially suitable for storing and transporting cell lines particularly sensitive to stress situations. In this regard, the system is adapted and especially suitable for transporting HEK-<NUM> cells, as well as Caco-<NUM> cells.

In order to use the system of the invention, firstly the cells must be immobilized on a support for transport. It may be used any type of support that allows cell transport. In a particular embodiment, the support is selected from a Petri dish, a multiwell plate, transwell membrane inserts, biomimetic systems, synthetic supports for 3D cultures, glass plates, slides or any type of support subjected to previous coating so as to facilitate cell adhesion, such as, e.g., a coating with collagen, polylysine or other similar elements.

When the cells reach their proper functional state and the proper confluence state is when they are ready to be coated with the system of the invention. In the context of the present invention, proper functional state is understood to be the state of the viable cells of the culture when they are capable of carrying out their assigned function for the assay for which they are intended. Once they reach said proper functional state and the proper confluence, the system of the invention is added, guaranteeing, thanks to the gelatin, that the cells will be perfectly immobilized and will maintain their functional state until they reach their final destination. Gelatin limits and reduces the mechanical stress to which the cells are subjected during the transport process.

Thanks to the joint action of the transport medium that reduces physiological stress, as well as gelatin that reduces the mechanical stress of the cells, it is produced a synergistic effect that provides the appropriate transport conditions for the cells to reach their destination in its optimal functional state.

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

A second aspect of the present invention relates to a method for the in vitro storage and/or transport of cell lines comprising:.

In a particular embodiment, the method comprises using:.

The first stage of the method (stage A) is aimed at preparing and accommodating the cell culture to be transferred. To this end, prior to coating the cells with the system of the invention, they are sown at the density determined for each cell type. In accordance with the type of assay for which they are intended, the cell lines will be sown on one type of support or another. The culture must be maintained for as long as necessary until it reaches the suitable functional and confluence, preferably changing the medium every <NUM>-<NUM> hours if necessary.

Although any suitable support for cell transport can be used, in particular the support used is selected from a Petri dish, a multiwell plate, transwell membrane inserts, biomimetic systems, synthetic supports for 3D cultures, glass plates, slides or any type of support previously coated so as to facilitate cell adhesion such as a coating with collagen, polylysine or other similar elements.

Once reached the suitable functional state, the cell line is coated with the system of the invention, comprising the transport medium and the gelatin solution that must be in the liquid state, in accordance with stage A of the method. The gelatin solution is prepared by dissolving gelatin in the transport medium, which acts as a solvent. Thanks to the use of the transport medium as a solvent, it is achieved that the cell line stored and / or transported according to the method of the present invention preserves the functional properties of the culture so that it can be used immediately once it has reached its final destination.

In stage B), the gelatin matrix is solidified at a temperature between <NUM> and <NUM>, whereupon the cells are immobilized and stabilized. The gelatin layer represents a physical protection for the cells and allows them to better withstand the mechanical stress resulting from the transport process.

Transport (stage C) is carried out at a temperature between <NUM> and <NUM> and allows transits greater than <NUM> hours, preferably greater than <NUM> hours and preferably up to <NUM> hours.

Once reached their destination and at the moment the immobilized culture is to be used, the plate is incubated with solid gelatin inside a cell incubator until it is completely liquefied, preferably at <NUM>, with <NUM> % humidity and <NUM> % CO<NUM> for <NUM> to <NUM> hours. Then, it is removed by aspiration and the specific culture medium for the cells in question is applied and the cells are incubated at <NUM> with <NUM> % humidity <NUM> % CO<NUM> until use.

Although the method of the present invention is applicable to any type of cell line, it is especially suitable for storing and/or transporting cell lines selected from HEK-<NUM> and Caco-<NUM>.

In another aspect, the present invention relates to a kit for the in vitro transport and storage of cell lines comprising:.

The cell storage and transport system of the kit of the invention comprises both the transport medium and the gelatin matrix in the terms and parameters described above.

Regarding the support, the kit can include any suitable support for cell transport, although in a preferred embodiment the support used is selected from a Petri dish, a multiwell plate, transwell membrane inserts, biomimetic systems, synthetic supports for 3D cultures, glass plates, slides or any type of support previously coated so as to facilitate cell adhesion, such as a coating with collagen, polylysine or other similar elements.

The kit of the present invention is in principle useful for transporting any type of cell line, although it is especially suitable for storing and/or transporting cell lines selected from HEK-<NUM> and Caco-<NUM> cell lines.

A last aspect of the invention is represented by the use of the system of the invention.

Although the system of the invention allows, in principle, the storage and / or transport of any cell line, it has been specially designed and developed for the transport of cell lines that are most sensitive to stress situations.

Preferably, the system of the present invention is useful for the storage and/or transport of HEK-<NUM> and Caco-<NUM> cell lines.

It has been observed that the system of the invention allows maintaining the cell viability of <NUM> % of the cells for <NUM> hours and of at least <NUM> % of the cells after <NUM> hours or more depending on the cell type. Therefore, the system of the invention allows the shipment and transport of cells and particularly sensitive cells such as HEK-<NUM> and Caco-<NUM> to practically any place on any continent.

The following examples serve to illustrate the invention, but are not intended to limit the scope of the invention in any way.

A series of assays were carried out on mock HEK-<NUM> and HEK-<NUM> MATE1 cells to test cell viability after <NUM> hours (<NUM> days) and <NUM> hours (<NUM> days) using different transport means. As will be seen, in this comparative study the improved survival effect of the transport composition of the invention was demonstrated against the maintenance medium commonly used for this type of cells (initial transport medium).

The following formulations were assayed:.

The comparative study was carried out on cells. Both the mock HEK-<NUM> and HEK-<NUM> MATE1 cells were sown at a density of <NUM>,<NUM> cells/well in a Poly-D-Lysine-coated <NUM>-well plate. <NUM> hours after seeding, having reached total confluence, the medium was removed and <NUM>µl/well of transport medium were added. The plate was left to temper inside the flow cabin, sealed with parafilm and then placed in the climatic chamber at <NUM>.

Plates were liquefied after exposure times to the transport medium of <NUM> and <NUM> hours. The transport medium was liquefied by introducing the plates at <NUM> in the CO<NUM> incubator for <NUM> minutes. Once the gelatin was liquefied, the transport medium was removed and HEK-<NUM> maintenance medium was applied (<NUM>µl/well).

After <NUM> hours, cell viability was assayed using Alamar Blue (<NUM>µl/well of <NUM> % Alamar Blue in a HEK-<NUM> maintenance medium, <NUM> hours of incubation in CO<NUM> incubator).

The percentage of cell viability was calculated with respect to the net values of RFU (Relative Fluorescence Units) of control cells at time zero prior to the application of the transport medium (TM): <MAT>.

Significant differences between groups were verified using Student's t-test (unpaired). Values of p≤<NUM> were considered statistically significant. Tables <NUM> and <NUM> below show the results of the comparative assays for both mock HEK-<NUM> and HEK-<NUM> MATE1 cells. The results can also be observed in <FIG> (mock HEK-<NUM>) and <FIG> (HEK-<NUM> MATE1).

The results with mock HEK-<NUM> cells demonstrate already at <NUM> hours that the differences between using a Leibovitz L15 basal medium versus DMEM D6046 without the presence of HEPES buffer provides a clear improvement in survival. This difference is minimized when HEPES is added to DMEM D6046 medium but not to Leibovitz L15 basal medium. However, once HEPES is added to the L15 medium, it is observed that the differences with the DMEM D6046 medium are very significant. A significant difference is also observed in the comparison between the L15 medium with or without HEPES. This indicates the importance of this buffer in cell survival. However, taken as a whole, these results suggest that the combination of Leibovitz L-<NUM> basal medium and HEPES buffer is of vital importance to the survival of mock HEK-<NUM> cells after <NUM> hours.

At <NUM> hours, the results were similar, although the differences are accentuated, such that the use of Leibovitz L-<NUM> basal medium with <NUM>% HEPES increases cell viability in an extremely significant way compared to the other controls.

The results in HEK-<NUM> MATE1 cells after <NUM> hours are similar to the results in mock HEK-<NUM> cells, but it is observed that the impact of HEPES on survival is more significant in all comparisons.

At <NUM> hours, a similar pattern is observed, although what can be inferred is that the relative importance of L-<NUM> basal medium to the survival of HEK-<NUM> MATE1 cells is more significant in the system of the invention than for mock HEK-<NUM> cells. This does not imply that HEPES is unimportant in survival after <NUM> hours, but again it suggests the synergistic effect between the use of Leibovitz L-<NUM> basal medium together with HEPES buffer in significantly improving cell viability after both <NUM> hours and <NUM> hours in HEK-<NUM> MATE1 cells and in mock HEK-<NUM> cells.

In conclusion, the L-<NUM>+<NUM> % HEPES composition assayed allows cell transport at room temperature, keeping cell viability substantially unchanged for up to <NUM> days.

After <NUM> days of exposure to room temperature, the transport medium based on L15 + <NUM>% HEPES maintains a cell viability of <NUM>% and <NUM>% of HEK-<NUM> mock and MATE1, respectively.

A series of assays were carried out to assess the integrity of the cell layer in Caco2 cells at <NUM> days (<NUM> hours) and <NUM> days (<NUM> hours) using different means of transport. The currently available mean of transport allows journeys for a maximum period of <NUM> days, which is why it was sought to test whether the transport system of the present invention could overcome this time barrier. As will be seen in this comparative study, the improved survival effect of the transport composition of the invention was demonstrated against the current mean. For this, the cell integrity of the monolayer was evaluated after the test period by measuring the transepithelial resistance of the cell monolayer (TEER).

The following equipment was used to carry out the test:.

The comparative study was carried out on Caco2 cells, and following the manufacturing procedure stipulated internally for the CacoReady <NUM>-wells product.

Cell seeding was performed at a density of <NUM>,<NUM> cells / well in <NUM><NUM>-well plates (# <NUM>, Corning). The cultures were maintained for <NUM> days, proceeding with changes of medium every <NUM> hours approximately.

On day <NUM> after sowing, having reached total confluence, the medium was withdrawn and <NUM>µL was added in the apical compartment and <NUM>µL in the basal compartment of transport medium. The plates were allowed to warm inside the flow cabinet, sealed with parafilm and subsequently placed in the climatic chamber at <NUM>.

The plates were liquefied after exposure times to the transport medium of <NUM> days (day <NUM>) and <NUM> days (day <NUM>). The transport medium was liquefied by placing the plates at <NUM> ° C in the CO2 incubator for <NUM> hours. Once the transport medium was liquefied, it was removed and Caco2 maintenance medium (<NUM>µL / apical + <NUM>µL / basal) was applied.

<NUM> hours after liquefaction, medium changes are made every <NUM> days until day <NUM> and <NUM> of the process, in which the transepithelial resistance of the cell monolayer (TEER) is randomly measured to check the integrity of the cell barrier.

The transepithelial resistance is calculated relative to the net TEER values by the membrane area of the seed well.

The results obtained on the transepithelial resistance of the cell monolayers for the current transport medium (SM CR) as well as for the transport system of the present invention (SM L15) are detailed in Table <NUM> for the periods of <NUM> days and <NUM> days respectively. These results are also detailed graphically in <FIG>.

As can be seen, the current means of transport (SM CR) does not allow the integrity of the monolayer to be maintained for a period of <NUM> days since the TEER results are outside the established limit specification.

Claim 1:
A system for the in vitro storage and transport of cell lines comprising:
a) a cell transport medium comprising:
i. <NUM> % to <NUM> % by volume of Leibovitz L-<NUM> basal medium
ii. <NUM> % to <NUM> % by volume of FBS
iii. <NUM> % to <NUM> % by volume of HEPES
iv. <NUM> % to <NUM> % by volume of Glutamine
v. <NUM> % to <NUM> % by volume of a mixture of penicillin and streptomycin
b) a solidified matrix of <NUM>-<NUM> of gelatin per ml of transport medium.