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 method of controlling the tetrahydrocannabinol content of a tissue culture, the method comprising the steps of: a) selecting Cannabis sativa leaf tissue for culture; and b) growing a tissue culture for between 10-28 days from the selected cannabis sativa leaf tissue in a liquid based medium comprising Murashige and Skoog basal powdered medium and a callus induction media while controlling the ultraviolet light exposure of the tissue culture to control the tetrahydrocannabinol content of the tissue culture, wherein the ultraviolet light is controlled during growth of the tissue culture such that the tissue culture is exposed to ultraviolet light of an intensity greater or equal to 1200 lumens but less than 2000 lumens and the ultraviolet light exposure is cycled through alternating periods of exposure and darkness and wherein the light exposure is controlled such that tissue culture is constantly exposed to photosynthetically active radiation to provide at least 0.5 moles of photons per day, wherein each period of exposure is at least 30 minutes and each period of darkness is at least 30 minutes, wherein the carbon dioxide content of the environment in which the tissue culture is grown is controlled to increase tissue growth, wherein the selected cannabis sativa leaf tissue of step a) was previously grown in a liquid based medium while controlling the light exposure of the tissue culture to control the tetrahydrocannabinol content of the tissue culture and wherein the tissue culture is exposured to ultraviolet B light, consisting of light of wavelength 280-315 nm which results in increased production of tetrahydrocannabinol and callus formation begins after about one month of culturing.

Description

EXAMPLE

i) Liquid Media

(1) Starting Media

(2) 0.44% Murashige and Skoog basal powdered medium

(3) 1.0% NAA (naphthalene acetic acid) 0.004% stock solution

(4) 3.0% sucrose

(5) Distilled water to 100%

(6) Equipment

(7) Glass bottle with cap

(8) Magnetic stirrer

(9) Sterile plastic plant culture dishes

(10) Glass pipettes

(11) pH meter

(12) Autoclave

(13) Laminar flow cabinet

(14) Balance

(15) Nescofilm

(16) Phytagel

(17) 1M NaOH solution

(18) 0.1M NaOH solution

(19) The liquid media was prepared in the following manner:

(20) 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);

(21) b) The media was then pH adjusted to pH 5.75 and solidified with 0.2% phytagel;

(22) c) The media was then autoclaved for 20 minutes at 121° C. and then poured into sterile plastic plant tissue culture dishes.

ii) Culture Initiation

(23) Reagents

(24) Liquid media (as prepared in the manner set out above)

(25) Cannabis sativa leaf tissue

(26) Equipment

(27) Sterile glass beakers

(28) Sterile distilled water

(29) Sterile scalpel

(30) Sterile tweezers

(31) 10% bleach solution

(32) 70% ethanol solution

(33) 1M NaOH solution

(34) 0.1M NaOH solution

(35) The culture was initiated in the following manner:

(36) 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;

(37) b) The leaf tissue was then washed three times with sterile (autoclaved) distilled water;

(38) c) The sterile washed leaf tissue was asceptically cut into disc shapes in a sterile laminar flow cabinets;

(39) d) The leaf tissue slices were placed onto the prepared plates containing callus induction media, and plates were sealed with Nescofilm®.

(40) e) The plates were placed in the dark at 27° C. and callus formation began to appear after about 1 month.

iii) Media Preparation for Cultures

(41) Reagents

(42) 3% sucrose

(43) 0.44% Murashige and Skoog basal powdered medium

(44) 1% naphthalene acetic acid (NAA) 0.004% stock solution

(45) 0.01% vitamin solution (0.05% pyridoalhydrochlorid, 0.1% thiamine dichloride, and

(46) 0.05%g nicotinic acid)

(47) 1M NaOH solution

(48) 0.1M NaOH solution

(49) Distilled water to 100%

(50) Equipment

(51) 1L glass bottle

(52) Magnetic stirrer

(53) 20×250 m conical

(54) 20 sheets of foil approximately 20 cm×20 cm

(55) Glass pipettes

(56) pH meters

(57) Autoclave

(58) Laminar flow cabinet

(59) Balance

(60) The media was prepared in the following manner:

(61) 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;

(62) b) Mix using a magnetic stirrer until all dry components dissolved, then pH adjust with 1M and 0.1 M NaOH to a pH of 5.75;

(63) 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;

(64) d) Immediately following sterilization place flasks in laminar flow cabinet and allow to cool to ambient temperature.

iv) Inoculation and Subculture of Established Cultures

(65) Reagents

(66) Friable callus

(67) 70% ethanol

(68) Equipment

(69) Laminar flow cabinet

(70) Bunsen burner

(71) Prepared media

(72) 20 sterile sheets of foil approximately 20 cm×20 cm

(73) Several pairs of tweezers or small forceps

(74) Wide spatulas with holes

(75) Broad spectrum PAR lighting

(76) UVA and UVB lighting

(77) The inoculation and subculture of established cultures was carried out in the following manner:

(78) a) Sterilize inside of laminar flow cabinet with 70% ethanol;

(79) b) Sterilize all tweezers and spatulas by dipping in 70% ethanol, then flaming till red hot. Allow to cool inside laminar flow cabinet;

(80) c) Remove foil from prepared media flask;

(81) 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);

(82) e) Flame the neck of the flask and cover with a sterile sheet of foil;

(83) 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;

(84) g) Remove foil from prepared media flask;

(85) h) Remove foil from flask containing dispersed cell suspension cultures (produced by inoculation at point f);

(86) i) Take wide spatula with holes, sterilize, allow to cool, and scoop out the cells.

(87) Add these cells to the fresh media. Aim to add approximately 5 g tissue to 50 ml of media;

(88) j) Flame the neck of the flask and cover with a sterile sheet of foil;

(89) 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

(90) l) After 14 days use the cell suspension culture for further subcultures or harvest cells.

(91) Lighting Regime 1

(92) Constant exposure to PAR at a rate of 0.5 moles of photons per day; and

(93) Constant exposure to UVB and UVA radiation at an intensity of approximately 500 lumens.

(94) Lighting Regime 2

(95) Constant exposure to PAR at a rate of 0.5 moles of photons per day; and

(96) 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.