METHOD OF PRODUCTION OF PHYTOCANNABINOIDS FOR USE IN MEDICAL TREATMENTS

Abstract

A method of producing cannabinoids for use in medical treatments by growing cultured Cannabis sativa plant cells through tissue culture, the method comprising the steps of: selecting Cannabis sativa leaf tissue for culture; and growing a tissue culture from the selected leaf tissue in a liquid based medium whilst controlling the light exposure of the tissue culture to control the cannabinoid content of the tissue culture. Control of the light exposure can enable the phytocannabinoid content of the grown tissue culture to be tailored to the use intended for the tissue culture. For example, the THC content of the tissue culture can be controlled to be maximised or minimised depending on the intended use. Use of tissue culture is beneficial as compared to prior art methods as it allows for genetic consistency and reduces the resources necessary to produce plant cells containing phytocannabinoids.

Claims

1. A Cannabis cell suspension culture comprising: a. a liquid based plant cell culture medium; and b. Cannabis callus cells.

2. The cell culture of claim 1, further comprising cannabinoids, other phytoconstituents, or any combination thereof.

3. The cell culture of claim 2, wherein the cell suspension culture is grown under controlled UV light exposure and wherein the cell suspension culture further comprises a controlled content of cannabinoids, a controlled content of other phytoconstituents, or any combination thereof.

4. The cell culture of claim 2, wherein the concentration of cannabinoids in the culture ranges from about 1% (g/mL) to about 60% (g/mL).

5. The cell culture of claim 2, wherein the cannabinoids comprise THC and CBD in a ratio of about 1:1 to about 10:1.

6. The cell culture of claim 2, wherein the cell suspension culture is about 10 to about 28 days-old.

7. The cell culture of claim 2, wherein the cell suspension culture comprises Cannabis cells at a density ranging from about 10,000 cells/mL to about 100,000 cells/mL.

8. The cell culture of claim 1, wherein the Cannabis cell suspension culture comprises liquid cell culture medium inoculated with callus cells grown on solid cell culture media.

9. The cell culture of claim 8, wherein the solid media cell culture is about 15 to about 45 days-old.

10. The cell culture of claim 8, wherein the liquid cell culture medium comprises about 0.5 to about 1.5 g/mL or about 1 g/mL of the Cannabis callus cells.

11. The cell culture of claim 8, wherein the solid media cell culture is grown under a full spectrum of photosynthetically active radiation (PAR) and wherein the solid media cell culture is grown under minimized exposure to UV radiation.

12. The cell culture of claim 11, wherein the concentration of cannabinoids ranges from about 0.05% to about 2%.

13. The cell culture of claim 11, wherein the density of Cannabis cells in the cell suspension culture ranges from about 10,000 to about 100,000 cells/mL.

14. The cell culture of claim 11, wherein the solid media cell culture is about 10 to about 28 days-old.

15. The cell culture of claim 1, wherein the Cannabis cell suspension culture comprises liquid cell culture medium inoculated with callus cells grown in liquid cell culture media.

16. The cell culture of claim 1, wherein the cell suspension culture comprises a volume ranging from about 1 mL to about 60 mLs, from about 40 mL to about 100 mLs, or from about 90 mLs to about 3 Ls or more.

17. The cell culture of claim 1, wherein the cell suspension culture comprises a volume ranging from about 100 Ls to about 5000 Ls or more.

18. The cell culture of claim 1, further comprising a cell culture container or a bioreactor comprising the cell suspension culture.

19. The cell culture of claim 1, wherein the cell culture medium comprises naphthalene acetic acid (NAA).

20. The cell culture of claim 1, wherein the liquid cell culture medium comprises water, macronutrients, micronutrients, vitamins, amino acids or nitrogen supplements, carbon sources, organic supplements, growth regulators, or any combination thereof.

21. The cell culture of claim 20, wherein the liquid cell culture medium comprises NAA, cannabinoids, other phytoconstituents, or any combination thereof.

22. The cell culture of claim 1, wherein the liquid cell culture medium is Anderson rhododendron medium, CHU(N6) medium, CLC/Ipomoea medium, Chee & Pool (C2D) vitis medium, De greef & jacobs medium, DKW/JUNGLANS medium, Eriksson(er) medium, Gamborg B5 medium, Gresshof & doy (DBM2) medium, Hellers medium, kao michayluk medium, knudson corchid medium, Lindemann Orchid medium, Litvay medium, Linsmaier & Skoog medium, McCowns woody plant medium, Murashige & Skoog medium, Murashige & Miller medium, nitsch medium, NLN medium, orchimax medium, quoirin & Lepoivre medium, rugini olive medium, schenk & hildebrandt medium, S-Medium, vacin and went medium, white medium, westvaco WV3 medium, and any combination thereof, or any modification thereof.

23. The cell culture of claim 1, wherein the Cannabis callus cells are derived from Cannabis leaves, flower buds, flower cells, bract cells, trichomes, or any combination thereof.

24. The cell culture of claim 1, wherein the Cannabis cell suspension culture is comprised in conditions for storage.

25. The cell culture of claim 1, wherein the Cannabis callus cells are derived from Cannabis leaves.

26. The cell culture of claim 25, wherein the leaves are selected to provide the correct phytocannabinoid composition

27. A Cannabis cell suspension culture comprising: a. a liquid based plant cell culture medium; b. a cell culture container or bioreactor comprising the cell culture medium; and c. Cannabis callus cells.

28. The cell culture of claim 27, further comprising cannabinoids, other phytoconstituents, or any combination thereof.

29. A Cannabis cell suspension culture comprising: a. a liquid based plant cell culture medium comprising naphthalene acetic acid (NAA); b. Cannabis callus cells; and c. cannabinoids, other phytoconstituents, or any combination thereof.

30. The cell culture of claim 29, wherein the cell suspension culture is derived from a subculture of Cannabis callus cells.

31. The cell culture of claim 29, wherein the cell suspension culture is about 10 to about 28 days-old.

32. The cell culture of claim 29, wherein the cell suspension culture is about 15 to about 45 days-old.

33. A solid media Cannabis cell culture comprising: a. a solid-based plant cell culture medium comprising NAA; and b. Cannabis callus cells.

34. The solid media Cannabis cell culture of claim 33, wherein the solid media Cannabis cell culture is about 15 to about 45 days-old.

35. The solid media Cannabis cell culture of claim 33, wherein the solid media cell culture is grown under minimized exposure to UV radiation.

36. A kit for preparing a Cannabis cell suspension culture of claim 1, the kit comprising: a. a plant cell culture medium; b. Cannabis callus cells; and c. optionally a cell culture container or a bioreactor.

37. The kit of claim 36, wherein the plant cell culture medium is a liquid based culture medium, a solid culture medium, or both.

38. The kit of claim 36, wherein the cell culture container comprises the Cannabis callus cells.

Description

EXAMPLE

[0033] i) Liquid Media

[0034] Starting Media

[0035] 0.44% Murashige and Skoog basal powdered medium

[0036] 1.0% NAA (naphthalene acetic acid) 0.004% stock solution

[0037] 3.0% sucrose

[0038] Distilled water to 100%

[0039] Equipment

[0040] Glass bottle with cap

[0041] Magnetic stirrer

[0042] Sterile plastic plant culture dishes

[0043] Glass pipettes

[0044] pH meter

[0045] Autoclave

[0046] Laminar flow cabinet

[0047] Balance

[0048] Nescofilm

[0049] Phytagel

[0050] 1M NaOH solution

[0051] 0.1M NaOH solution

[0052] The liquid media was prepared in the following manner: [0053] a) The starting media was Murashige and Skoog (MS) media with 3% sucrose and 1% naphthalene acetic acid (from concentrated stock solution of 0.004% w/v); [0054] b) The media was then pH adjusted to pH 5.75 and solidified with 0.2% phytagel; [0055] c) The media was then autoclaved for 20 minutes at 121° C. and then poured into sterile plastic plant tissue culture dishes.

[0056] ii) Culture Initiation

[0057] Reagents

[0058] Liquid media (as prepared in the manner set out above)

[0059] Cannabis sativa leaf tissue

[0060] Equipment

[0061] Sterile glass beakers

[0062] Sterile distilled water

[0063] Sterile scalpel Sterile tweezers

[0064] 10% bleach solution

[0065] 70% ethanol solution

[0066] 1M NaOH solution

[0067] 0.1M NaOH solution

[0068] The culture was initiated in the following manner: [0069] a) The leaf tissue of Cannabis Sativa was sterilised by immersion in 70% ethanol for 2 minutes, followed by immersion in 10% bleach solution for 10 minutes; [0070] b) The leaf tissue was then washed three times with sterile (autoclaved) distilled water; [0071] c) The sterile washed leaf tissue was asceptically cut into disc shapes in a sterile laminar flow cabinet; [0072] d) The leaf tissue slices were placed onto the prepared plates containing callus induction media, and plates were sealed with Nescofilm. [0073] e) The plates were placed in the dark at 27° C. and callus formation began to appear after about 1 month.

[0074] iii) Media Preparation for Cultures

[0075] Reagents

[0076] 3% sucrose

[0077] 0.44% Murashige and Skoog basal powdered medium

[0078] 1% naphthalene acetic acid (NAA) 0.004% stock solution

[0079] 0.01% vitamin solution (0.05% pyridoalhydrochlorid, 0.1% thiamine dichloride, and

[0080] 0.05% g nicotinic acid)

[0081] 1M NaOH solution

[0082] 0.1M NaOH solution

[0083] Distilled water to 100%

[0084] Equipment

[0085] 1 L glass bottle

[0086] Magnetic stirrer

[0087] 20×250 m conical

[0088] 20 sheets of foil approximately 20 cm×20 cm

[0089] Glass pipettes

[0090] pH meters

[0091] Autoclave

[0092] Laminar flow cabinet

[0093] Balance

[0094] The media was prepared in the following manner: [0095] a) Mix 3% sucrose, 0.44% Murashige and Skoog basal powder, 1% NAA stock, and 0.01% vitamin solution and prepare to 100% with distilled water; [0096] b) Mix using a magnetic stirrer until all dry components dissolved, then pH adjust with 1M and 0.1M NaOH to a pH of 5.75; [0097] c) Take 20×250 ml conical flasks, to each add 50 ml media and seal neck of flask with foil; sterilize in autoclave at 121° C., 103 kPa for 25 minutes; [0098] d) Immediately following sterilization place flasks in laminar flow cabinet and allow to cool to ambient temperature.

[0099] iv) Inoculation and subculture of established cultures

[0100] Reagents

[0101] Friable callus

[0102] 70% ethanol

[0103] Equipment

[0104] Laminar flow cabinet

[0105] Bunsen burner

[0106] Prepared media

[0107] 20 sterile sheets of foil approximately 20 cm×20 cm

[0108] Several pairs of tweezers or small forceps

[0109] Wide spatulas with holes

[0110] Broad spectrum PAR lighting

[0111] UVA and UVB lighting

[0112] The inoculation and subculture of established cultures was carried out in the following manner: [0113] a) Sterilize inside of laminar flow cabinet with 70% ethanol; [0114] b) Sterilize all tweezers and spatulas by dipping in 70% ethanol, then flaming till red hot. Allow to cool inside laminar flow cabinet; [0115] c) Remove foil from prepared media flask; [0116] d) Take sterilized tweezers and remove thumbnail sized pieces of friable callus from the plant tissue. Break up into finely dispersed cells and add to flask. Aim to add approximately 5 g of tissue to 50 ml media (10% w/v); [0117] e) Flame the neck of the flask and cover with a sterile sheet of foil; [0118] f) Place the flask on a shaker at 120 rpm, in a dark room heated to 27° C. Leave until a thick dispersed cell suspension culture can be observed, approximately 2 weeks, or until the density of cells in the culture ranges from about 10,000 cells/mL to about 100,000 cells/mL, from about 20,000 cells/mL to about 90,000 cells/mL, from about 30,000 cells/mL to about 80,000 cells/mL, from about 40,000 cells/mL to about 70,000 cells/mL, from about 50,000 cells/mL to about 60,000 cells/mL, from about 10,000 cells/mL to about 60,000 cells/mL, from about 20,000 cells/mL to about 70,000 cells/mL, from about 30,000 cells/mL to about 80,000 cells/mL, from about 40,000 cells/mL to about 90,000 cells/mL, or from about 50,000 cells/mL to about 100,000 cells/mL; [0119] g) Remove foil from prepared media flask; [0120] h) Remove foil from flask containing dispersed cell suspension cultures (produced by inoculation at point f); [0121] i) Take wide spatula with holes, sterilize, allow to cool, and scoop out the cells. Add these cells to the fresh media. Aim to add approximately 5 g tissue to 50 ml of media; [0122] j) Flame the neck of the flask and cover with a sterile sheet of foil; [0123] k) Place the flask on a shaker at 120 rpm in, subject to one of the two lighting regimes set out below, and heated to 27° C. for 14 days; and [0124] l) After 14 days, or until the density of cells in the culture ranges from about 10,000 cells/mL to about 100,000 cells/mL, from about 20,000 cells/mL to about 90,000 cells/mL, from about 30,000 cells/mL to about 80,000 cells/mL, from about 40,000 cells/mL to about 70,000 cells/mL, from about 50,000 cells/mL to about 60,000 cells/mL, from about 10,000 cells/mL to about 60,000 cells/mL, from about 20,000 cells/mL to about 70,000 cells/mL, from about 30,000 cells/mL to about 80,000 cells/mL, from about 40,000 cells/mL to about 90,000 cells/mL, or from about 50,000 cells/mL to about 100,000 cells/mL, use the cell suspension culture for further subcultures or harvest cells.

[0125] Lighting Regime 1

[0126] Constant exposure to PAR at a rate of 0.5 moles of photons per day; and

[0127] Constant exposure to UVB and UVA radiation at an intensity of approximately 500 lumens. In some aspects, the concentration of cannabinoids using this lighting regime ranges from about 0.01% (g/mL) to about 5% (g/mL), from about 0.05% (g/mL) to about 4% (g/mL), from about 0.1% to about 3% (g/mL), from about 0.5% (g/mL) to about 4% (g/mL), from about 0.1% (g/mL) to about 2% (g/mL), from about 0.5% (g/mL) to about 1%. (g/mL), from about 0.05% (g/mL) to about 1%. (g/mL), from about 0.1% (g/mL) to about 1.5%. (g/mL), or from about 1% (g/mL) to about 2%. (g/mL).

[0128] Lighting Regime 2

[0129] Constant exposure to PAR at a rate of 0.5 moles of photons per day; and Periodic exposure to UVB and UVA radiation at an intensity of approximately 1500 lumens, the periodic exposure consisting of alternating 1 hour periods of exposure and 1 periods in which there is no UVB and UVA exposure. In some aspects, the concentration of cannabinoids using this lighting regime ranges from about 1% (g/mL) to about 60% (g/mL), from about 5% (g/mL) to about 50% (g/mL), from about 10% (g/mL) to about 40% (g/mL), from about 20% (g/mL) to about 40% (g/mL), from about 40% (g/mL) to about 60% (g/mL), from about 1% (g/mL) to about 50% (g/mL), or from about 20% (g/mL) to about 50% (g/mL).