METHOD FOR PRODUCING MODIFIED MESENCHYMAL STROMAL STEM CELLS WITH IMPROVED PROPERTIES, MODIFIED CELLS OBTAINED BY THIS METHOD, COMPOSITION INCLUDING SUCH CELLS

Abstract

The present invention relates to a non-viral method for the production of modified mesenchymal stem/stromal cells comprising the use of an additive rich in human-derived platelet growth factors, derived from platelet concentrates, aimed to promote the expansion of said cells. The method further comprises the phase of submitting the cells to electroporation and suspending said mixture in the growth-medium enriched in human-derived platelet growth factors and keeping in incubation. In this way, it is possible to achieve a chemical-physical, non-viral method in compliance with the Good Manufacturing Practice Guidelines (GMP) for advanced cell therapy products, allowing transiently or stably transfect/deliver gene constructs based on electric pulses administration to cells. Said production method is particularly simple and advantageous in terms of productivity and cost-effectiveness, when compared to methods involving viral vectors.

Claims

1. A method for the modification of mesenchymal stromal stem cells comprising the following phases: a) seeding said cells on a plastic support in the presence of an appropriate growth-medium promoting their expansion; b) adding at least one medium additive rich in human-derived platelet growth factors aimed to promote the expansion of said cells; c) detaching said expanded cells from the plastic surface; d) adding to said expanded cells a gene construct in order to obtain a mixture; characterized in that said method also comprises the following phases: e) submitting said mixture to one or more electrical pulses to electroporate said mixture; f) suspending said electroporated mixture having been submitted to one or more electrical pulses in said growth-medium comprising the additive rich in human-derived platelet growth factors, and transferring the resulting mixture into a suitable container for incubation; g) resuspending said cell mixture adding an amount of growth-medium containing a percentage of additive rich in human-derived platelet growth factors higher than the same growth-medium used in the previous phase (b); h) transferring the modified cells to a plastic support for cell culture and incubating; i) at the end of the incubation, adding to said support a volume of growth-medium devoid of said additive rich in human-derived platelet growth factors to restore the equivalent additive concentration applied in the growth-medium used in said phase (b) for cell expansion.

2. The method according to claim 1 wherein said mesenchymal stromal stem cells are isolated from adipose tissue or other sources such as adult tissues and fetal annexes selected from the group consisting of bone marrow, dental pulp, umbilical cord, Wharton's jelly, and amniotic fluid.

3. The method according to claim 1, wherein said additive rich in human-derived platelet growth factors is obtained from whole blood or platelet-apheresis plasma procedures.

4. The method according to claim 1, wherein said additive rich in human-derived platelet growth factors used in phase b) is added in percentages comprised within a range of 1% to 20% vol/vol, 1.25% to 15% vol/vol, 2.5% to 12% vol/vol, 5% vol/vol.

5. The method according to claim 1, wherein said electrical pulses are of different magnitude and waveform capable of creating a plurality of transient pores in the cell membranes through which said gene construct may permeate the cell.

6. The method according to claim 1, wherein in said phase f) the mixture of cells expanded by means of the growth-medium containing the additive rich in human-derived platelet growth factors is incubated at about 37 C. for a period of less than 60 minutes.

7. The method according to claim 1. wherein said additive rich in human-derived platelet growth factors used in phase g) is added at percentages that are about twice as high as the percentages of the same additive used in phase b) above.

8. Mesenchymal stromal stem cells expanded by a growth-medium containing an additive rich in platelet growth factors, electroporated and genetically modified, and obtained by the method of claim 1.

9. The use of mesenchymal stromal stem cells expanded by a growth-medium containing an additive rich in human-derived platelet growth factors according to claim 8 for cytotoxic treatment against a tumor mass.

10. The use of mesenchymal stromal stem cells expanded by mean of a growth-medium containing an additive rich in human-derived platelet growth factors and processed according to claim 8 to induce expression of trophic, anti-inflammatory, and/or antioxidant factors promoting tissue regeneration/reconstruction.

11. The use of mesenchymal stromal stem cells expanded by a growth-medium containing an additive rich in human-derived platelet growth factors and processed according to claim 8 for the treatment of pulmonary fibrosarcoma and/or glioblastoma.

12. A composition comprising mesenchymal stromal stem cells expanded by a growth-medium containing an additive rich in human-derived-platelet growth factors and processed according to claim 8.

13. The method according to claim 1, wherein in said phase f) the mixture of cells expanded by means of the growth-medium containing the additive rich in human-derived platelet growth factors is incubated at about 37 C. for about 20 minutes.

14. The method according to claim 1, wherein said additive rich in human-derived platelet growth factors used in phase g) is added at percentages that are around 10% vol/vol.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0060] The following detailed description refers to a preferred embodiment of a method of the present invention.

EXAMPLE 1 OF THE INVENTION

[0061] 5010.sup.3 mesenchymal stem/stromal cells isolated from adipose tissue (ASC) were seeded in T-75 tissue culture flask (75 cm.sup.2) (BD Biosciences; Franklin Lakes, NJ, US) in 15 ml of growth-medium of the type Minimum Essential Medium Eagle-Alpha Modification (-MEM) (Lonza; Basel, Switzerland).

[0062] In other embodiments of the present invention it is possible to use other commercially available formulations specifically dedicated to the expansion of mesenchymal cells or of adhesion growing fibroblastoid cells. The number of T-75 flask or different sizes can be selected, based on the required final number of cells to be transfected.

[0063] Cells were added with 5% (vol/vol) additive rich of human-derived platelet growth factors (SRGF) extracted from platelets collected by plasma-platelet apheresis.

[0064] The cell expansion procedures involving SRGF addition were performed in aseptic conditions under a Class II laminar flow safety cabinet (sterile cabinet).

[0065] Expanded cells were placed in a 5% CO2 incubator at 37 C. for at least 3 days. Cells can be left in the incubator without medium exchange or, if necessary, a half volume medium change can be performed before the end of the incubation time.

[0066] Cells were collected before contiguous cell-cell contact (confluence) was achieved, as this could limit transfection efficiency.

[0067] ASC were detached from plastic surface, after double wash with phosphate buffered saline (PBS), by adding 1 ml of undiluted trypsin-ethylenediaminetetraacetic acid 10 at 37 C. (TrypLe Select; Life Technologies-Thermo Fisher Scientific, Waltham, MA, USA).

[0068] ASC containing flasks were placed back in the incubator at 37 C. for 2 minutes until cells were detached, as assessed by microscope observation.

[0069] Trypsin activity was neutralized adding 4 ml of complete growth medium (SRGF-containing) and the whole cell mixture was collected.

[0070] Cells were concentrated using a standard bench centrifuge (1200 RPM for 5 minutes). Thereafter, cells were counted by a manual hemacytometer (Burker chamber) or by a validated automated device. For each transfection procedure 50010.sup.3 cells were collected and cells were concentrated by using the standard bench centrifuge (1200 RPM for 5 minutes).

[0071] Cells were resuspended in NF buffer (Ingenio Electroporation Solution, Mims Bio Corporation, Madison; USA) (50010.sup.3 cells in 100 l buffer).

[0072] It was added to the cell's mixture 2 g of the expression vector encoding for the green fluorescent proteinGFP (even if other vectors and genes can be selected in other embodiments); the concentration of the original expression vector stock solution was within the range of 0.5-1 g/L in pure water. The mixture was prepared taking advantage of high quality plasmid kit and purification procedures.

[0073] The cell mixture was then transferred into the appropriate electroporation cuvette (Ingenio) where it was subjected to electric pulse administration by the C17 program, using the Nucleofector 2b device (Lonza).

[0074] Immediately after electroporation phase, 0.5 ml of complete medium were added into the electroporation cuvette. Using the appropriate pipette (Ingenio), cells were transferred to a clean centrifugation tube where they were incubated at 37 C. for 20 minutes.

[0075] Cells were then resuspended in the centrifuge tube by gentle pipetting and divided in two containers (wells) of a 6-well plate (BD) previously filled with 2 ml of warm complete medium (37 C.), with twice the concentration (10% vol/vol) of supernatant containing high amounts of growth factors (SRGF) than the concentration (5% vol/vol) of the same SRGF used to expand the cells.

[0076] Cells were then placed in a 5% CO.sub.2 incubator at 37 C. for nearly 1 hour.

[0077] At the end of the incubation, an equal volume of growth medium without additive rich of human-derived platelet growth factors (SRGF) was added to equilibrate the final growth factor concentration at the optimal condition for cell expansion (e.g., relatively to the addition of 2 ml of complete medium containing 10% SRGF for cell resuspension, 2 ml of growth medium without SRGF were added to reconstitute 5% SRGF concentration in a final volume of approximately 4 ml).

[0078] After approximately 12-48 hour from transfection, cell viability, gene expression and transfection efficiency were evaluated.

EXAMPLE 2 OF THE INVENTION

[0079] Identical to the above-mentioned Example 1, with the only difference that cells were expanded in 1.25% vol/vol medium additive rich of human-derived platelet growth factors SRGF (instead of 5%, as in the Example 1).

[0080] Moreover, as a consequence, after electroporation step, cells were resuspended in the centrifuge tube using a doubled SRGF concentration (i.e. 2.5% vol/vol, instead of 10%, as in the Example 1).

EXAMPLE 3 OF THE INVENTION

[0081] Identical to the above-mentioned Example 1, with the only difference that cells were expanded in 2.5% vol/vol medium additive rich of human-derived platelet growth factors SRGF (instead of 5%, as in the example 1).

[0082] Moreover, as a consequence, after electroporation step, cells were resuspended in the centrifuge tube using a doubled SRGF concentration (i.e. 5% vol/vol, instead of 10%, as in the Example 1).

EXAMPLE 4 OF THE INVENTION

[0083] Identical to the above-mentioned Example 1, with the only difference that cells were expanded in 10% vol/vol medium additive rich of human-derived platelet growth factors SRGF (instead of 5%, as in the example 1).

[0084] Moreover, as a consequence, after electroporation step, cells were resuspended in the centrifuge tube using a doubled SRGF concentration (i.e. 20% vol/vol, instead of 10%, as in the Example 1).

EXAMPLE 5 OF THE INVENTION

[0085] Identical to the above-mentioned Example 1, with the only difference that cells were expanded in 20% vol/vol medium additive rich of human-derived platelet growth factors SRGF (instead of 5%, as in the example 1).

[0086] Moreover, as a consequence, after electroporation step, cells were resuspended in the centrifuge tube using a doubled SRGF concentration (i.e. 40% vol/vol, instead of 10%, as in the Example 1).

EXAMPLE 6 (CONTROL)

[0087] Identical to the above-mentioned Example 1, with the only difference that cells were expanded without medium additive rich of human-derived platelet growth factors (SRGF); instead 10% vol/vol fetal bovine serum (FBS) was added. Moreover, upon detachment, expanded ASC were placed in the incubator for 4 minutes at 37 C. (instead of 2 minutes, i.e. the incubation time to detach 5% SRGF expanded ASCsee example 1 of the invention). Finally, cells were resuspended in the tube, still using a doubled FBS concentration (i.e. 20% vol/vol, instead of 10% FBS, as during the cell expansion phase) in analogy with the Example 1 of the present invention, where SRGF concentration was doubled when compared to the cell expansion phase.

Evaluation

[0088] Cell samples collected for the present invention, as described in examples 1-5 and in the control example number 6, were analyzed for viability, gene construct expression and transfection efficiency. Viability of transfected cells was comprised between 772% (using the C-17 program of the Nucleofector 2b (Lonza)) and 593% (when cells were expanded in presence of 10% FBS). In contrast, the viability of cells expanded in FBS and transfected by both programmes was less than 5%. The following table reports transfection efficiency obtained using the C-17 program on ASC expanded in presence of different concentrations of SRGF (medium additive rich of human-derived platelet growth factors) or FBS (control medium additive).

TABLE-US-00001 TABLE 1 Transfection efficiency Additive (Mean S.E.M.) Sample 1 (invention) 5% SRGF 50 6% Sample 2 (invention) 1.25% SRGF 32 3% Sample 3 (invention) 2.5% SRGF 35 7% Sample 4 (invention) 10% SRGF 48 4% Sample 5 (invention) 20% SRGF 50 3% Sample 6 (control) 10% FBS 22 5%

[0089] From the data shown in Table 1, it can be seen that the samples of the invention in which ASC were expanded in presence of SRGF (medium additive rich of intraplatelet growth factors) from 1.25 to 20% vol/vol were characterized by an increased electroporation efficiency when compared to the control samples, where cells were expanded without SRGF but in presence of 10% vol/vol fetal bovine serum (FBS). Cells expanded in presence of 5% SRGF showed the highest electroporation efficiency, equal to the efficiency showed by cells expanded in presence of 10% and 20% of the same additive. Cells expanded with the 2.5% and 1.25% additive showed a reduction in electroporation efficiency, but still remaining superior to the control sample.

[0090] Addition of 5% SRGF was shown to be an optimal and safe condition to expand mesenchymal stem/stromal cells derived from adipose tissue (ASC).

[0091] Naturally, many modifications and variations of the described preferred embodiments will be evident to those skilled in the art, still remaining within the scope of the invention.

[0092] Therefore, the present invention is not limited to the preferred embodiments described, illustrated only by way of non-limiting example, but is defined by the following claims.