METHOD FOR PRODUCING CELL FLAPS

Abstract

The present invention refers to an in vitro method for producing a flap of genetically modified cells on fibrin substrate and to the flap so obtained.

Claims

1. An in vitro method for producing a flap of genetically modified cells on a fibrin substrate, comprising: a) plating feeder cells on the upper surface of a fibrin substrate so as to obtain a fibrin substrate on which said feeder cells are adhered; b) plating and cultivating to subconfluence said genetically modified cells on said fibrin substrate onto which feeder cells are adhered, said fibrin substrate being positioned on a solid support so that the genetically modified cells do not interact with the surface of said support so as to obtain a flap of genetically modified cells adhered to said fibrin substrate; and c) detaching the flap of genetically modified cells adhered to said fibrin substrate from the support in a form similar to a sheet to obtain a flap of genetically modified cells on said fibrin substrate.

2. The method according to claim 1, wherein the feeder cells are plated on the fibrin substrate from 2 to 24 hours before plating the genetically modified cells.

3. The method according to claim 1, further comprising: before step c), the steps: b) removing the culture medium and/or b) washing the flap of genetically modified cells adhered to said fibrin substrate with a washing solution and/or after step c), the step of: d) placing the obtained flap of genetically modified cells on fibrin substrate in a transport container.

4. The method according to claim 1, wherein the fibrin substrate has dimensions of from about 0.32 cm.sup.2 to about 300 cm.sup.2.

5. The method according to claim 1, wherein the fibrin substrate comprises from about 20 to about 100 mg/ml of fibrinogen and from about 1 to about 10 IU/ml of thrombin.

6. The method according to claim 5, wherein the fibrin substrate comprises from about 20 to about 50 mg/ml of fibrinogen, and from about 3 to about 8 IU/ml of thrombin.

7. The method according to claim 6, wherein the fibrin substrate comprises from about 20 to about 25 mg/ml of fibrinogen and from about 2 to about 4 IU/ml of thrombin.

8. The method according to claim 7, wherein the fibrin substrate comprises about 23.1 mg/ml of fibrinogen and about 3.1 IU/ml of thrombin.

9. The method according to claim 1, wherein said genetically modified cells are epithelial cells.

10. The method according to claim 9 wherein said genetically modified cells are epidermal cells.

11. The method according to claim 9 wherein said genetically modified cells are keratinocytes.

12. The method according to claim 1, wherein said genetically modified cells have been transduced with a gene or a cDNA selected from the group consisting of: a) at least one chain selected from the group consisting of: beta-3, 3 and 2 chain of laminin-5, and/or b) collagen 17 and/or c) at least one 64 integrin and/or d) collagen 7 and/or e) keratin 5 and Keratin 14 and/or f) Plectin.

13. The method according to claim 1, wherein said genetically modified cells have been transduced with a gene or a cDNA selected from the group consisting of: beta-3 chain of laminin 5, collagen 7 and collagen 17.

14. A flap of genetically modified cells on a fibrin substrate, obtainable by the method of claim 1.

15. A flap of genetically modified cells on a fibrin substrate, wherein said genetically modified cells are epithelial cells.

16. The flap of genetically modified cells on a fibrin substrate according to claim 15 wherein said genetically modified cells are epidermal cells.

17. The flap of genetically modified cells on a fibrin substrate according to claim 16 wherein said cells are keratinocytes.

18. The flap according to claim 14 wherein the genetically modified cells are transduced with a gene or a cDNA selected from the group consisting of: a) at least one chain selected from the group consisting of: beta-3, 3 and 2 chain of laminin-5, and/or b) collagen 17 and/or c) at least one 64 integrin and/or d) collagen 7 and/or e) keratin 5 and Keratin 14 and/or f) Plectin.

19. The flap according to claim 14 wherein the genetically modified cells are transduced with a gene or a cDNA selected from the group consisting of: beta-3 chain of laminin 5, collagen 7 and collagen 17.

20. The flap according to claim 14 wherein the genetically modified cells are cells that have been transduced with a retroviral vector.

21. The flap according to claim 14 wherein the fibrin substrate comprises from about 20 to about 100 mg/ml of fibrinogen and from about 1 to about 10 IU/ml of thrombin.

22. The flap according to claim 21 wherein the fibrin substrate comprises from about 20 to about 50 mg/ml of fibrinogen, and from about 3 to about 8 IU/ml of thrombin.

23. The flap according to claim 22 wherein the fibrin substrate comprises from about 20 to about 25 mg/ml of fibrinogen and from about 2 to about 4 IU/ml of thrombin.

24. The flap according to claim 23 wherein the fibrin substrate comprises about 23.1 mg/ml of fibrinogen and about 3.1 IU/ml of thrombin.

25-29. (canceled)

Description

[0117] The invention will be now illustrated by means of non-limiting examples referring to the following figures.

[0118] FIG. 1. Representative pictures of cultured keratinocytes grown on plastic. The image on the right is representative of the flap prior to detachment and assembly for transport.

[0119] FIG. 2. Representative images of the flap detachment with Dispase II and two preparations of the flap originated from plastic. The center image shows a flap not conforming to the release due to the presence of air bubbles, while the photo on the right represents the image of a flap conforming to the release.

[0120] FIG. 3. Representative images of the confluences reached by growing keratinocytes on fibrin supports at the time of detachment and preparation for transport.

[0121] FIG. 4. Representative images of the preparation of the genetically modified epidermis flap.

EXAMPLES

[0122] Materials and Methods

[0123] Isolation of Epidermal Keratinocytes from Biopsy of Human Skin

[0124] Primary human keratinocytes are isolated from 2-9 cm.sup.2 skin biopsies after submission and adhesion to informed consent. The biopsy is subjected to enzymatic digestion in Trypsin-EDTA solution at 37 C. To obtain maximum yield and minimize the risk of toxicity from exposure to trypsin-EDTA or prolonged suspension time of keratinocytes during extraction, till 6 sequential trypsinizations of 30 each are performed. After each trypsinization the cellular material is recovered and the trypsinizations 1-3 and 4-6 are combined and plated in plastic supports according to the cell yield at a density of 1.3310.sup.4 cells/cm.sup.2 on a feeder layer of lethal irradiated murine cells 3T3-J2 (Rheinwald, J. et al. 1975). The medium used consists of a mixture of Dulbecco's modified Eagle (DMEM) and Ham F12 (2:1) supplemented with 10% fetal bovine serum, 0.5% penicillin-streptomycin, 2% glutamine, insulin (5 g/ml), adenine (0.18 mM), hydrocortisone (0.4 g/ml), cholera toxin (0.1 nM) and triiodothyronine (2 nM). After 3 days, this culture medium is removed and replaced with KC (KNO medium containing 10 ng/ml of EGF) which is changed every other day until the subconfluence stage is reached.

[0125] Primary Culture and Transduction of Keratinocytes.

[0126] After reaching the subconfluence (80-95%), the cells are subjected to enzymatic digestion with trypsin-EDTA for 15 at 37 C. The cell suspension thus obtained is plated to a density of 1.3310.sup.4 cells/cm.sup.2 on the feeder layer composed of 3T3J2 and AM12 cells (packaging cells) (Mavilio et al., Nature, 2006) producing the retroviral vector carrying the beta3 chain of laminin 5 (810.sup.4 cells/cm.sup.2) in a 1:2 ratio. After 3 days, the cells are transferred onto a new 3T3-J2 layer feeder, which is plated on a support of the same size and grown to subconfluence. Once the transduced cells have reached subconfluence, they are trypsinized as previously described. The cells thus obtained are divided into appropriate freezing vials of 1-210.sup.6 cells. In parallel, process control tests are performed. Process controls consist of:

[0127] 1. Evaluation of the number of transduced cells. A number from 1000 to 10,000 cells is plated on feeder layer in multiwells/chambers for the evaluation of the number of positive cells at the transgene.

[0128] 2. Evaluation of the colony forming efficiency CFE. A number from 500 to 2000 cells is plated on a 100 mm dish and stained after 12 days in rhodamine.

[0129] 3. Evaluation of the number of 3T3J2 and AM12 cells present in the cell suspension.

[0130] Gel Fibrin Production in 144cm.sup.2 Supports

[0131] Fibrin is produced by the inventor and comprises two fibrinogen reagents (23.1 mg/ml) and thrombin (3.1 IU/ml) produced by Kedrion and sold with the commercial name Kolfib. The production process involves three phases:

[0132] 1. Preparation of fibrinogen solution and thrombin

[0133] 2. Preparation of fibrin support

[0134] 3. Fibrin compliance test

[0135] 1. A thrombin (kedrion) vial containing 625 IU or 1250 IU of thrombin is reconstituted in 10 ml of buffer consisting of NaCl (1.1%) and CaCl2 (1 mM). The entire content is then transferred to a 50 ML tube to which other 10 ML buffer will be added. If the starting vial contained 625 UI of thrombin, a 1:5 dilution of the reconstituted solution is made, e.g. 5 ML of the reconstituted solution are transferred in a new 50 ml tube and after 20 ML of buffer are added. If the starting vial contained 1250 UI of thrombin, a dilution of 1:10 of the reconstituted solution is made, e.g 18 ML of the reconstituted solution are transferred in a new 50 ML tube and other 162 ml of buffer are added. The solution is prepared at room temperature and examined to ensure that there are no solubilized thrombin solutions. A 120 mg or 240 mg fibrinogen vial is solubilized in 2.59 ML or 5.184 ML of buffer containing NaCl (1.73%) and CaCl2 (1.23 mM) and aprotinin (3574 KIU/ml). The reconstituted solution is incubated at 36.5 C. for 30 to 60 minutes to complete the solubilization.

[0136] 2. The fibrin gel (or fibrin substrate) is prepared in a 144 cm.sup.2 support in untreated plates for cell culture. To obtain a 100 mm thick gel, 6 ML of thrombin solution and 6 ML of fibrinogen solution are mixed to obtain a homogeneous mixture. The plates thus prepared are left at room temperature for 10-15 min until full polymerization and then stored at 4 C. for up to one month.

[0137] 3. Before releasing the fibrin gel are subjected to compliance checks (see Table 3).

[0138] Flaps Preparation

[0139] The first step in the preparation of the genetically modified epidermis flap consists in plating 3T3-J2 feeder cells on the 144 cm.sup.2 fibrin support. The feeder cells are plated 2 to 24 h before the keratinocyte plating or at the same time of the thawing of transduced keratinocytes.

[0140] The feeder cells are plated on the upper surface of the fibrin, the homogeneous plating of the feeder is controlled through an optical microscope inspection with 10 and 20 magnification. A vial of transduced cells is thawed and plated on supports previously described at a density of 500,000-3,000,000 cells transduced for a surface of 144 cm.sup.2. The prepared culture is transferred to an incubator at 37 C., 6% CO2 and 99% humidity, monitored and the medium is changed every two days until reaching the subconfluence (7-14 days).

[0141] Preparation of Flaps for Transport

[0142] Once the subconfluence is reached, the culture medium is removed. The genetically modified epidermis flap is washed three times with a washing and transport solution consisting of DMEM and L-Glutamine. Once the washings have been carried out, the flap is detached from the plastic container by means of sterile forceps and then transferred to the transport container for the flap to which the transport medium will be added. The container is then sealed to keep the environment sterile inside it. At this stage it will be necessary to avoid the formation of air bubbles. The fibrin flap thus prepared has a stability of 36 hours.

REFERENCES

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