A METHOD OF CULTURING CELLS

Abstract

A method of culturing autogenous cells as well as a method of treating a mammalian patient is described. The cells are cultured on a substrate material having a surface treated with a fatty acid ester so as to have a strong hydrophobic surface and autogenous plasma obtained from a blood sample of the patient is used as a growth medium in order to culture the cells. The substrate material is also used as a transfer dressing for transferring the cultured cells to the patient and as a wound cover dressing. The invention also describes a kit as well as a system for culturing autogenous cells using the culture method.

Claims

1. A method of culturing autogenous cells for use in the treatment of a mammalian patient, the steps including: a. Providing a substrate material having a surface treated with a fatty acid ester so as to have a strong hydrophobic surface; b. Placing cells obtained from the patient on the hydrophobic surface of the substrate material, so as to culture autogenous cells thereon; and c. Applying autogenous plasma obtained from a blood sample of the patient to the cells as a growth medium in order to culture the cells.

2. The method as claimed in claim 1 wherein the substrate material is used as a transfer dressing for transferring the cultured cells to the patient.

3. The method as claimed in either claim 1 or claim 2 wherein the substrate material is further used as a wound cover dressing.

4. The method of claim 1 wherein the substrate material comprises a cellulose acetate, cotton gauze or nonwoven fabric and the fatty acid is selected from a dialkyl carbamoyl chloride (DACC) and an alkyl ketene dimer (AKD).

5. The method as claimed in claim 4 wherein the DACC is dihexadecyl-carbamoyl chloride or dioctadecyl-carbamoyl chloride.

6. The method of claim 1 wherein the cells are selected from skin cells or epithelial cells which include fibroblasts and melanocytes.

7. The method of claim 1 wherein the plasma is platelet rich plasma (PRP).

8. The method of claim 1 which includes a further step of moisturizing the cells using a hydrogel, at least every 2 to 4 days.

9. A method of treating a mammalian patient by transplanting autogenous cells, cultured in vitro, including the steps of: a. Obtaining cells from the patient in a biopsy; b. Providing a substrate material having a surface treated with a fatty acid ester so as to have a strong hydrophobic surface; c. Placing the cells on the substrate material and culturing autogenous cells thereon, using autogenous plasma obtained from a blood sample of the patient as a growth medium in order to culture the cells; d. Transplanting the autogenous cultured cells onto the patient, using the substrate material as a transfer dressing and wound cover dressing.

10. A kit for culturing autogenous cells for use in the treatment of a mammalian patient, the kit including: a. a Trypsin solution or solution precursors for separating epidermal and dermal cells of a biopsy obtained from a patient b. a substrate material having a surface treated with a fatty acid ester so as to have a strong hydrophobic surface and onto which the cells may be placed so to culture autogenous cells thereon; and c. a plurality of tubes for containing plasma obtained from a blood sample of the patient.

11. The kit as claimed in claim 10 which further comprises a plurality of syringes for extracting plasma from a blood sample obtained from the patient; a plurality of sterile needles and 15/0 blades (theatre stock) and a tube of hydrogel.

12. The kit as claimed in claim 11 in the form of a partitioned container with a plurality of zones; including a first zone containing the trypsin solution or solution precursors; a second zone containing the substrate material and optionally a supporting frame for holding the substrate material in a flattened condition; a third zone containing the tubes; and a fourth zone containing the syringes, sterile needles and blades and hydrogel; and wherein each zone is individually sealed by a peel away film cover.

13. The kit of claim 10 which further includes a package insert containing instructions for using the kit for culturing autogenous cells.

14. A system for culturing autogenous cells for use in the treatment of a mammalian patient, the system including: a. an incubator having a plurality of substrate zones, each zone being associated with a patient and being configured to receive a substrate material having cells obtained from the associated patient placed thereon so as to culture autogenous cells thereon; b. an identifying component for identifying the patient associated with the substrate zone; and, c. an applicator unit for operatively applying, as a growth medium in order to culture cells, autogenous plasma obtained from a blood sample of the patient to cells on a substrate material contained in the substrate zone associated with the patient.

15. The system as claimed in claim 14, wherein the applicator unit includes a robotic arm operable to select a dosage unit associated with the patient, the dosage unit containing the autogenous plasma obtained from a blood sample of the patient, and wherein the applicator unit applies the autogenous plasma contained in the selected dosage unit.

16. The system as claimed in claim 15, wherein the system includes a dosage unit bank including a plurality of dosage unit zones each of which being configured to hold a dosage unit, each dosage unit zone being associated with a patient, wherein the identifying component is operable to identify a dosage unit zone associated with the patient and wherein the robotic arm is operable to select and extract a dosage unit from the identified dosage unit zone.

17. The system as claimed in claim 16, wherein the system includes a plurality of dosage units, each of which includes an outlet and a plunger, and wherein the robotic arm is operable to position the outlet proximate the substrate zone associated with the patient and to urge the plunger towards the outlet to operatively apply autogenous plasma contained therein to cells on a substrate material contained within the substrate zone.

18. The system of claim 14, wherein the incubator is temperature controlled, and wherein the temperature selected from the range of 30 degrees Celsius to 38 degrees Celsius is maintained.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0054] In the drawings:

[0055] FIG. 1 is an illustration of one embodiment of a kit for culturing autogenous cells for use in the treatment of a mammalian patient; and

[0056] FIG. 2 is a schematic diagram which illustrates an exemplary system for culturing autogenous cells for use in the treatment of a mammalian patient.

DETAILED DESCRIPTION OF THE INVENTION

[0057] A method of culturing autogenous cells as well as a method of treating a mammalian patient is described. The cells are cultured on a substrate material having a surface treated with a fatty acid ester so as to have a strong hydrophobic surface and autogenous plasma obtained from a blood sample of the patient is used as a growth medium in order to culture the cells. The substrate material is also used as a transfer dressing for transferring the cultured cells to the patient and as a wound cover dressing. A kit as well as a system for culturing autogenous cells using the culture method is also described.

[0058] The cells were grown on a low cost hydrophobic substrate material, in this embodiment the hydrophobic dressing known as CUTIMED SORBACT?, which served as a growth template for epithelial cells and well as a transfer medium and a wound cover dressing.

[0059] Animal products, such as bovine plasma which is commonly used as growth medium for culturing cells, were not used in the culturing process of the present invention. Instead a patient's autogenous plasma, extracted from a blood sample of the patient, was used as a growth medium to feed the cells daily. The cells were also shown to proliferate equally well in sterile and in non-sterile conditions.

[0060] Aspects of the invention will now be described in more detail with reference to the following non-limiting examples.

EXAMPLES

[0061] Materials

[0062] Equipment: [0063] Two incubators, a centrifuge, acid-citrate-dextrose (ACD) tubes for platelet rich plasma, a sterile instrument pack from a burns theatre, sterile gloves, 20 ml syringes, 18G and 16G needles, CUTIMED SORBACT? dressing pads and ribbon gauze, sterile green cloths, face masks, sterile theatre clothes, shoe covers and head covers, 15/0 blades (theatre stock), a light microscope, slides, fixative and loupes (3.5? magnification 45 cm focus).

[0064] Biological requirements: [0065] Autogenous (patient's own) skin biopsy: 7?3 cm left inguinal, autogenous blood for platelet rich plasma (PRP) (36 ml blood in 6 ml ACD tube yielded 22-25 ml of PRP) and trypsin (enzyme from beef pancreas to separate epithelial cells from dermal elements, from an anatomy laboratory).

[0066] Method

[0067] The incubator was cleaned with a chlorhexidine solution and an alcohol solution and sterile gauze with sterile water and set to warm up to 36.8 degrees Celsius. The target temperature was 37 degrees Celsius.

[0068] A skin biopsy was transported from theatre to laboratory in a sterile specimen container, immediately after an operation to remove the skin was performed. With loupes on and using the scalpel with a number 15/0 blade, the epidermis and dermis was cut off and separated. Small fragments of epithelium were cut smaller. Some keratinocytes were scraped off without using loupes.

[0069] Boiling water was used to sterilise two specimen bottles which were used to mix Trypsin powder in sterile water. The small fragments of epithelial skin were then placed in the sterile specimen bottles with the trypsin solution to further separate the epidermal and dermal elements.

[0070] A RECELL kit was also sterilised with boiling water and used to assist in cell preparation. A small sift in the kit was used to rinse off the trypsin, and forceps were then used to pick up minute specks of epithelial cells and place them on six hydrophobic CUTIMED SORBACT? pads in the central area of the pads. Other cells were placed in the two sterilised specimen bottles.

[0071] Six tubes of blood with 6 ml in each were then centrifuged at 3500 revolutions per minutes (rpm) for 8 minutes. Thereafter the tops of the tubes were removed and the plasma was drawn up with a sterile 20 ml syringe and 18-gauge needle. Meticulous attention was given to avoiding drawing up any red blood cells which might contain pyrogenic cytokines which are pro-inflammatory, like Interleukin-1, 6 and tumour necrosis factor-alpha (TNF-alpha).

[0072] The plasma was sprayed onto the cells on the CUTIMED SORBACT? pads and into the specimen bottles containing cells. CUTIMED SORBACT? ribbon gauze was laid down between the pads to form a border for the cell growth and to avoid the cells from growing over the edges of the pads.

[0073] The cells were nourished daily with fresh PRP. Moistness in the incubator was maintained by adding sterile water to the water inlet on the outside of the incubator on a daily basis. Intrasite gel was applied with a sterile glove over the cultured epithelial autograft (CEA) every third day to prevent the cells from drying out too much.

[0074] On day 14 of the CEA growth, the target day for cell transplantation was reached, exactly 108 days after the patient's admission.

[0075] The incubator with the cultured cells was taken to theatre and the cells kept at 37 degrees Celsius. After anaesthesia commencement, 36 ml of blood for PRP was taken from the patient's A-line. The patient was washed with chlorhexidine soap and draped with sterile clothes. Wound debridement was done by gentle scraping of raw areas with a metal ruler's blunt side/tip. This was done over previously grafted xenograft areas as well as a donor site on the left medial lower leg. Haemostasis was obtained with swabs soaked in an adrenalin solution (Ringers Lactate 1 litre and 1 ampule of adrenaline and 800 mg Lignocaine). CUTIMED SORBACT? pads with the CEA were then transplanted directly onto the wounds of the back, left arm, left thigh and knee with the simultaneous application of PRP. On the patient's right arm, thigh and leg, CUTIMED SORBACT? pads were used with squirted CEA and fresh PRP. A sacral bedsore was also treated with squirted CEA from cells grown in the two sterile specimen bottles, with autogenous PRP on CUTIMED SORBACT?pads. The back dressing were fixed with IOBAN, an iodine containing film dressing. The rest of the dressings were bandaged with cling/crepe.

[0076] Results:

[0077] A 78.16% graft take was achieved. The CEA included cells grown from deeper skin layers (not just epithelial cells as in the classic technique, but including fibroblasts and melanocytes).

[0078] Plasma Dilution Studies

TABLE-US-00001 Ratio of sterile water fluid gel to plasma obs vol gel/layer vol vol cell vol Cell/Fluid % Cell/Gel % 1:1 10 mm Fluid 10 mm 6 ml 0 ml 0.1 ml 1 0 0.75:1 10 mm + Fluid 10 mm 6 ml 0 ml 0.1 ml 1 0 0.5:1 15 mm + Gel 9 mm 0.4 ml 2 ml 0.6 ml 6.70 30 0.25:1 10 mm + Gel 7 mm 0.4 ml 3 ml 0.5 ml 7.14 16.67 undiluted 15 mm + Fluid 5 mm 4 ml 0 ml 0.17 ml 3.4 0 *uncontrolled 46 ml 0 46 ml 0 ml 0.6 ml 1.3 0 *Uncontrolled = original sample cells that were in a suspension of sterile water and Trypsin were kept in a plastic dish on a table and covered with a sterile green cloth with no temperature or humidity regulation. Plasma was added to the cells daily in small amounts. Cells grew in this uncontrolled suboptimal area at room temperature (more or less 24 degrees Celsius), and under non-sterile conditions.

[0079] Results of Plasma Dilution Studies [0080] 1. Cells appear to be robust and easy to grow with autogenous plasma. [0081] 2. Plasma dilution can be done safely up to a ratio of 0.5:1 (sterile water: plasma) with good effect, but best results are obtained by daily feeding the cells undiluted plasma. Practically every alternative day can be skipped and cells should still survive in suspension. [0082] 3. The preferred dressing is definitely hydrophobic and not hydrophilic. Any hydrogel-addition to the back of such dressings are not good for cell growth. [0083] 4. The dual function of acting as a growth medium and anti-bacterial agent makes Sorbact the best choice.

[0084] Cell Types

TABLE-US-00002 Cell types/ Epidermal Dermal Adnexal Central Membrane fibres layers layers Structures Cell Mass border keratinocytes, Dense cell Darker fibres, No Dense, less No fibroblasts masses Elastin at edges keratinocytes; Dense cell Darker fibres, No Dense, less Not clear fibroblasts, elastin masses Elastin at edges Fibrillar Less dense Dark and light No Denser at Yes, 1 thick Collagen/elastin cells fibrils edges layer Keratinocytes Outer Not sure, No Denser at Yes, 1 thick Other fibres border dense CM edges layer Tubular fibrils, other E cells in Multiple No Dense, less Not dark/ cells, Dermal between F streaky fibrils at edges clear keratinocytes, Yes Denser No Dense, less No dermal fibres masses fibres at edges Enc Gr. tree-like Not sure No Dense, Borders, keratinocytes controlled organized single Enc Gr. Yes Yes Possibly Dense, Yes, 4 Keratinocytes, ducts; border/epid layers dermal cells glands Enc Gr. Not seen Yes Yes Dense, Yes, 4 Keratinocytes, border/epid layers dermal cells Encapsulted cell yes, cell Not sure No Dense Not clear growth areas masses dark, shades Encapsulted areas + yes Yes, Muscle, Not sure Light Yes, 4 Tree pattern C & E, K coloured layers Encapsulated Yes, darker Yes, lighter, No Light Lighter grey, growth. K, E & C. areas. transparent coloured yellow Encapsulated Yes, darker Yes, lighter, Yes Light Yes, 4 growth. K, E & C. areas transparent coloured layers Encapsulated Yes, thicker Yes, light Yes. Sweat Light Dense Yes, 4 growth. K, E & C. outer m yellow; pink gland/hair organized layers Encapsulated Yes, thicker Yes, light, Yes, clear Light Dense Yes, 4 growth. K, E & C. outer m translucent gland, duct organized layers

[0085] Comparative Dressing Study

[0086] Dried out dressings: 3-4 months after CEA?3-4 days of Feeds. [0087] 1. Intrasite Conformable [0088] Skin can grow, but becomes interwoven/attached to the fibres from which it appears that it would be difficult to have tangential layers of skin cells forming. The fibres of the dressing are very coarse. [0089] 2. Biobrane [0090] Organised fibres and patterns of the dressing. No clear masses of epithelial cells seen. [0091] 3. Cutimed Sorbact Swabs [0092] Abundant cellular growth visible between darker fibres of the dressing. [0093] 4. Cutimed Sorbact Dressing Pads [0094] Upside down: Gauze at bottom: No cells seen on bottom surface. 4?magnification. [0095] Next older cells: Top side of cells: at 4?magnification multiple cells visible between the Sorbact fibres. [0096] Younger dressing 2 weeks old with CEA: at 4?magnification abundantly much more cells visible of increased density. Cells seen growing over the Sorbact fibres and in between them. [0097] 5. Sorbact Swabs: Two weeks old. [0098] Even though plasma was noted to seep through the dressing pad [0099] Sorbact gauze cells. More abundant growth with some cells appearing brownish, others grey. [0100] 6. Hydrogel pad [0101] Cells don't migrate well and there is pooling of plasma. [0102] 7. Cutimed Sorbact Hydro [0103] Pooling of plasma not good for lateral growth of the cells.

[0104] Findings: [0105] 1. Gauze type dressings are not good for growing monolayers of cells because the cells get stuck in the fibre structure and growth is haphazard. [0106] 2. Although cells may grow on Nylon mesh like Biobrane, the growth with the method of the present invention was not detectable. [0107] 3. Abundant cellular growth was seen with the Sorbact dressings. [0108] 4. The worst performing Sorbact for cell culture was where hydrogel pads was present, which led to pooling of plasma and therefore less lateral growth, and more vertical growth stimulation. [0109] 5. The hydrophobic properties appear to be very conducive to accelerate cell growth when compared to hydrophilic dressings: The hydrogel pad did not allow plasma to seep through and caused pooling which is bad for lateral cell growth. [0110] 6. The method described above using the Sorbact dressing pad (more rigid, less prone to deformity as a result of cell contractions) is probably the best, but the Sorbact gauze is less rigid and more prone to deformity by cells. [0111] 7. One would be able to grow cells on many surfaces, but there is none known that will give dual antibacterial and growth acceleration properties with the correct amount of moisture control for effective cell culture as Sorbact. [0112] 8. Other hydrogels (such as Cutimed gel BSN) other than Intrasite gel were tested, but the contents were found to be too watery. Intrasite gel appears to have the right thickness to act as an additional moisturizer to the cells.

[0113] Another aspect of the invention extends to a portable kit (1) for culturing autogenous cells for use in the treatment of a mammalian patient, as illustrated in one embodiment shown in FIG. 1. The kit (1) is in the form of a sterile, partitioned container (2) having a plurality of zones which include: [0114] a first zone (3) containing the trypsin solution or solution precursors (in the form of Trypsin powder and sterile water, which may be mixed to prepare a Trypsin solution), [0115] a second zone (5) containing the hydrophobic substrate material and a supporting frame for holding the substrate material in a flattened condition, [0116] a third zone (4) containing ACD tubes and [0117] a fourth zone (6) containing a plurality of syringes for extracting plasma from a blood sample obtained from the patient; a plurality of sterile needles, 15/0 blades (theatre stock), a tube of hydrogel and instructions for using the kit.

[0118] Each zone of the partitioned container (2) is individually sealed by a peel away film cover.

[0119] The Trypsin solution in the first zone (3) is used for separating epidermal and dermal cells of a biopsy obtained from a patient, as per the set of instructions contained in the fourth zone (6). The cells can be mixed with the solution directly in the first zone and the cells for culturing picked out of the zone (3) and placed onto the hydrophobic substrate material in the second zone (4).

[0120] Once a blood sample from a patient has been centrifuged and the PRP extracted, using syringes from the fourth zone (6), the plasma is placed in the ACD tubes, and can then be applied onto the cells as a growth medium on a daily basis. Application of the plasma can be performed according to set of instructions included in the fourth zone (6).

[0121] The instructions also include steps for moisturising the cells growing on the substrate material using the hydrogel, and for applying the hydrophobic substrate containing a layer of cells thereon onto a patient's wound.

[0122] Another aspect of the present invention extends to a system (100) for culturing autogenous cells for use in the treatment of a mammalian patient, one exemplary embodiment of which is illustrated in FIG. 2. The system (100) includes a controller (101), having processor (102) and a memory (104), and an applicator unit (106). Code executable by the processor (102) may be stored in the memory (104) or other non-transitory computer readable medium to enable the system (100) to perform various identification, control, and applicator functions, and the like.

[0123] The system (100) includes an incubator (110) having a plurality of substrate zones (112). Each zone (112) is configured to receive a substrate material (114) having cells obtained from a patient placed thereon. Placing cells of a patient on the substrate material (114) enables autogenous cells to be cultured thereon. The incubator (110) is temperature controlled such that a temperature selected from the range of 30 degrees Celsius to 38 degrees Celsius, and preferably 37 degrees Celsius, is maintained.

[0124] The system (100) also includes a dosage unit bank (120) which includes a plurality of dosage unit zones (122). Each dosage unit zone (122) is configured to hold a dosage unit (124) which is associated with a patient. Each dosage unit (124) includes a tubular body having an outlet at a first end thereof and a plunger at a second end thereof. A chamber is defined between the plunger and the outlet and contains autogenous plasma of the patient with which the dosage unit (124) is associated. In some embodiments, the dosage unit may be an acid-citrate-dextrose (ACD) dosage unit.

[0125] The system (100) includes an identifying component (130) which maintains a look-up table in which each substrate zone (112) is logically associated with coordinates (e.g. x-, y- and z-coordinates) and with a patient (e.g. a patient identifier such a patient name or patient number). In this manner, each substrate zone (112) is associated with a patient.

[0126] The identifying component (130) is operable to identify a patient associated with the substrate zone (112) or to identify a substrate zone (112) associated with a patient. In this embodiment, the identifying component (130) includes a tracking component (132) to track the position of the applicator unit (106) relative to stored coordinates of each substrate zone (112). The identifying component (130) identifies the patient associated with a substrate zone (112) by comparing the position of the applicator unit (106) to the coordinates of the substrate zones stored in the look-up table so as to identify the substrate zone to which the applicator unit (106) is proximate and to thereby identify the patient associated with the substrate zone (112).

[0127] The identifying component (130) further maintains a look-up table in which each dosage unit zone (122) is logically associated with coordinates (e.g. x-, y- and z-coordinates) and with a patient. In this manner, each dosage unit zone (122) is associated with a patient.

[0128] The identifying component (130) is operable to identify a dosage unit zone (122) associated with the patient. The tracking component (132) is operable to track the position of the applicator unit (106) relative to the coordinates of each dosage unit zone (122). The identifying component (130) is operable to identify the dosage unit zone (122) associated with the patient by comparing the position of the applicator unit (106) to the coordinates of the dosage unit zones (122) stored in the look-up table so as to identify the dosage unit zone (122) to which the applicator unit (106) is proximate and to thereby to identify the dosage unit zone associated (122) with the patient.

[0129] The applicator unit (106) includes a robotic arm (108) having an end effector (109) which is operable to select and extract a dosage unit (124) associated with a particular patient from the dosage unit zone (122) in which it is located and to apply autogenous plasma of the patient contained therein to cells on the substrate material (114) contained in the substrate zone (112) which is associated with the patient.

[0130] In use, a particular patient may be identified. The robotic arm (108) identifies a dosage unit zone (122) which is associated with the patient. The end effector (109) removes the dosage unit (124) from the identified dosage unit zone (122). The substrate zone (112) associated with the patient is then identified and the dosage unit (124) is positioned proximate the substrate material (114) located within the substrate zone (112) and having cells obtained from the patient placed thereon. The end effector (109) urges the plunger of the dosage unit (124) towards the outlet to apply the autogenous plasma contained therein to the cells placed on a substrate material (114). The autogenous plasma, when applied to the cells on the substrate material (114) acts as a growth medium in order to culture cells.

[0131] The system (100) is configured to operate autonomously such that cells for a number of patients may be cultured on a large scale and with minimal human intervention. For example, the applicator unit may run autonomously to apply autogenous plasma of a plurality of patients to their corresponding one or more substrate materials contained within the incubator so as to culture cells on a large and ongoing scale, for example for the duration that a patient is at a facility for treatment.

[0132] It should be appreciated that the embodiment of the system described above is exemplary and that various alterations and modifications may be made thereto. For example, in another embodiment, each substrate zone and dosage unit zone may have a computer-and/or human-readable identifier or indicia (e.g. a label and/or barcode) disposed thereon. The identifier may be usable by the identifying component to identify a patient associated with each of the substrate zones and dosage unit zones.

[0133] The hydrophobic dressing used for cell grafting in the present invention, CUTIMED SORBACT? which has a hydrophobic coating made from DACC, is capable of reducing the effects of shear stress, metabolic stress and tissue infection. The DACC attracts hydrophobic bacteria and renders them inert. The Applicant postulated and found that the hydrophobic action of CUTIMED SORBACT? would allow plasma, in the form of PRP, to be applied to the substrate and the cells could grow within the plasma droplets. Dressings of this type are further described in U.S. Pat. No. 4,617,326 and US patent application no. 2006/012980, which are incorporated herein by reference.

[0134] The applicant envisages that the methods and materials of the invention are particularly beneficial in avoiding infection due to the combined anti-infective effects of the hydrophobic CUTIMED SORBACT? dressing and the use of autogenous plasma. The applicant postulates that the use of autogenous plasma allows the anti-infective effects of any antibiotic treatment, tailored specifically to the patient's bacterial profile, to persist during the in vitro culturing process.

[0135] Throughout the specification and claims unless the contents requires otherwise the word comprise or variations such as comprises or comprising will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.