Cell suspension preparation technique and device
The present invention provides for methods and devices suitable for producing a transplantable cellular suspension of living tissue suitable for grafting to a patient. In applying the method and/or in using the device, donor tissue is harvested, subjected to a cell dissociation treatment, cells suitable for grafting back to a patient are collected and dispersed in a solution that is suitable for immediate dispersion over the recipient graft site.
1. A method for preparing a cell suspension for direct application to a patient in need of a treatment for a skin disorder or disease, which method comprises the steps of:
(a) subjecting a donor skin tissue sample comprising a dermis layer and an epidermis layer to a dissociating means comprising an enzyme to dissociate the dermis layer and the epidermis layer at a dermal-epidermal junction in the skin tissue sample, wherein the donor skin tissue sample provides an expansion ratio of 1:10 to 1:80 in the treatment;
(b) removing the donor skin tissue sample from the dissociating means used in step (a) and immersing the donor skin tissue sample in a reservoir of nutrient solution, wherein the nutrient solution is (i) free of serum xenogenic to the patient, (ii) capable of maintaining viability of the cells until applied to the patient and (iii) suitable for direct application to a recipient region on the patient;
(c) harvesting cells from a dermal surface facing the dermal-epidermal junction and an epidermal surface facing the dermal-epidermal junction in the reservoir of nutrient solution and suspending the harvested cells by mixing with the nutrient solution, thereby to produce a mixed suspension of dermal and epidermal cells in the reservoir of nutrient solution, wherein the harvested cells are suitable for grafting onto the patient and include keratinocyte basal cells, melanocytes and fibroblasts; and
(d) filtering the mixed suspension of dermal and epidermal cells from step (c) to remove cellular conglomerates greater than 200 μm, thereby producing a mixed cell suspension in the nutrient solution that is directly applied to the patient;
wherein the method does not comprise the steps of pelleting and resuspending the cells.
2. The method according to claim 1 wherein the enzyme is trypsin or trypsin-EDTA.
3. The method according to claim 1 wherein the enzyme is selected from the group consisting of dispase, collagenase, thermolysin, pronase, hyaluronidase, pancreatin, elastase and papain.
4. The method according to claim 1 wherein the skin tissue sample is subjected to the dissociating means for about 5 minutes to about 60 minutes.
5. The method according to claim 1 wherein the skin tissue sample is subjected to the dissociating means for about 15 minutes to about 20 minutes.
6. The method according to claim 1 wherein the nutrient solution is Hartmann's solution.
7. A method of treating a skin disorder or disease in a patient in need thereof, said method comprising the steps of:
(a) preparing the mixed cell suspension according to the method of claim 1 peri-operatively; and
(b) immediately administering the mixed cell suspension directly to the recipient region after said preparing step on the patient and without in vitro culturing.
8. A method for preparing a cellular suspension to provide a therapeutic preparation for direct application in a treatment of a skin disorder or disease, said method comprising:
(a) subjecting a donor skin tissue sample comprising a dermis layer and an epidermis layer to an enzyme to dissociate cohesive bonding between the dermis layer and the epidermis layer in the donor skin tissue sample, wherein the donor skin tissue sample provides an expansion ratio of 1:10 to 1:80 in the treatment;
(b) removing the donor skin tissue sample from a solution having the enzyme used in step (a) and immersing the skin tissue sample in a reservoir of nutrient solution, wherein the nutrient solution is (i) free of serum xenogenic to the patient, (ii) capable of maintaining viability of the cells until applied to the patient and (iii) suitable for direct application to a recipient region on the patient;
(c) harvesting cells from a dermal surface facing the dermal-epidermal junction and an epidermal surface facing the dermal-epidermal junction in the reservoir of nutrient solution and suspending the harvested cells by mixing with the nutrient solution, thereby to produce a mixed suspension of dermal and epidermal cells in the reservoir of nutrient solution, wherein the harvested cells are suitable for grafting onto the patient and include keratinocyte basal cells, melanocytes and fibroblasts; and
(d) filtering the mixed suspension of dermal and epidermal cells from step (c) to remove cellular conglomerates greater than 200 μm;
thereby producing a cellular suspension in the nutrient solution to provide a therapeutic preparation that is directly applied in the treatment of the skin disorder or disease.
9. The method according to claim 8 wherein the nutrient solution is Hartmann's solution.
10. The method according to claim 8 wherein the enzyme is selected from the group consisting of trypsin, trypsin-EDTA, dispase, collagenase, thermolysin, pronase, hyaluronidase, pancreatin, elastase and papain.
11. A method of treating a skin disorder or disease in a patient in need thereof, said method comprising the steps of:
(a) preparing the cellular suspension according to the method of claim 8 peri-operatively; and
(b) immediately administering the cellular suspension directly to the recipient region after said preparing step on the patient in need of treatment and without in vitro culturing.
12. The method of claim 1 , wherein the recipient region is receiving cell grafting.
13. The method of claim 1 , wherein the recipient region is receiving skin grafting.
14. The method of claim 8 , wherein the recipient region is receiving cell grafting.
15. The method of claim 8 , wherein the recipient region is receiving skin grafting.
16. A method for producing a mixed suspension of dermal and epidermal cells, the method comprising:
(a) providing a donor skin tissue sample in a reservoir of nutrient solution, the donor skin tissue sample being obtained from a patient and comprising a dermis layer, an epidermis layer, and a dermal-epidermal junction therebetween, wherein the donor skin tissue sample provides an expansion ratio of 1:10 to 1:80 in a skin treatment;
(b) harvesting cells from a dermal surface facing the dermal-epidermal junction and an epidermal surface facing the dermal-epidermal junction in the reservoir of nutrient solution and suspending the harvested cells by mixing with the nutrient solution, thereby to produce a mixed suspension of dermal and epidermal cells in the reservoir of nutrient solution; wherein prior to harvesting, the donor skin tissue sample has been subject to trypsin treatment; and
(c) filtering the mixed suspension of dermal and epidermal cells to remove cellular conglomerates greater than 200 μm,
wherein the nutrient solution is free of serum xenogenic to the patient and capable of maintaining viability of the dermal and epidermal cells, and wherein the filtered cells are directly applied to the patient for the skin treatment.
17. The method for preparing a cell suspension according to claim 1 wherein the dissociating means dissociates the dermis layer and epidermis layer by disrupting or weakening cohesive bonding between the dermis layer and the epidermis layer.
18. The method for preparing a cell suspension according to claim 1 , wherein step (b) further comprising rinsing the donor skin tissue sample after removal from the dissociating means and prior to immersing the donor skin tissue sample in the reservoir of nutrient solution.
19. A method of treating a skin disorder or disease in a patient, said method comprising the steps of:
(a) preparing the mixed suspension of dermal and epidermal cells according to the method of claim 16 peri-operatively; and
(b) immediately administering the mixed suspension of dermal and epidermal cells directly to a recipient site after said preparing step on the patient in need of treatment and without in vitro culturing.
20. The method of claim 1 , wherein the nutrient solution is a physiological saline.
21. The method of claim 8 , wherein the nutrient solution is a physiological saline.
22. The method of claim 8 , wherein the nutrient solution is a physiological saline.
23. The method of claim 1 , wherein the donor skin tissue sample is up to 2 cm×2 cm in size.
24. The method of claim 8 , wherein the donor skin tissue sample is up to 2 cm×2 cm in size.
25. The method of claim 16 , wherein the donor skin tissue sample is up to 2 cm×2 cm in size.