Brief introduction of Platform of CMC isolation and culture 榮雨生物科技有限公司 內部教育訓練教材

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1 Brief introduction of Platform of CMC isolation and culture 榮雨生物科技有限公司 內部教育訓練教材

2 1 What is CMC? It is the abbreviation for Cambial Meristematic Cells. Cambial meristematic cells are undifferentiated, and could be differentiated into all types of plant cells, so it is considered as PLANT STEM CELLS. Plant stem cells such as cambial meristematic cells locate ONLY in specific parts in the plant Root tip Cambium layer Leaf bud

3 2 Why should we study plants? 植物的應用極為廣泛 Because plants are raw materials with multiple use: crop food spices drink herbs vitamins Health food medicines The most important nutrition in the plant are phytochemicals (also known as 21th century vitamins), such as anthocyanins, lycopene, lutein, catechin, ginsenosides, resveratrol, etc. These active factors are why plant could be used as raw materials for herb, medicine and health food.

4 3 How to have enough plants or phytochemicals? 1 st Generation Field collection 2 nd Generation Farming 1,000,000 years age 10,000 years ago 18 th century 2005 now Fruits collection Vegetables collection Crop farming Vegetables farming Fruits farming But plants from field collection and farming are influenced by environment factors easily. These influences include plant yield, nutrition content and safety. Environment factors: climate, rainfall, bugs, heavy metal pollution, pesticides pollution, microbes, etc.

5 4 How to have enough plants or phytochemicals? 1 st Generation Field collection 2 nd Generation Farming 3 rd Generation Callus platform 1,000,000 years ago 10,000 years ago 18 th century 2005 now Fruits collection Vegetables collection Crop farming Vegetables farming Fruits farming Cultivation of orchid Crop genetic improvement Genetic modified organism Apple stem cells Rose stem cells Plant tissue culture technology was invented near 200 years age. People could culture plant CALLUS in the lab, and differentiate them into new plants. So it prevents the yield of phytochemicals from influence of environment factors.

6 5 Instability of callus limit its developments xylem (wood) Cambium phloem cortex plant tissue section dedifferentiation callus Callus cell culture or new plant (Differentiation) Callus cells (also named as dedifferentiated cells, DDCs) have many innate defects, and theses restrict applications of the callus technology. - Genetic variability, it leads to genetic mutation or make long term culture impossible - Slow growth, it leads to large-scale culture impossibility - Low phytochemicals yield, it leads to low business value and potential

7 6 How to have enough plants or phytochemicals? 1 st Generation Field collection 2 nd Generation Farming 3 rd Generation Callus platform 4 th Generation CMC platform 1,000,000 years ago 10,000 years ago 18 th century 2005 now Fruits collection Vegetables collection Grain farming Vegetables farming Fruits farming Cultivation of orchid Crop genetic improvement Genetic modified organism Apple stem cells Rose stem cells Yew CMC Wild ginseng CMC Ginkgo CMC Tomato CMC Cambial meristematic cells isolation and culture technology platform ( also known as CMC PLATFORM) was developed successfully by PLANT STEM CELL INSTITUTE (PSCI), Korea in It is the first time that people could isolate intact CMCs from a plant and culture them in the lab over 160 years.

8 7 BEST and FIRST scientific achievement in the world xylem (wood) Cambium phloem cortex plant tissue section peeling patented technology CMC & callus CMC culture or new plant (differentiation) Analyzed by Professor Gary Loake, institute of molecular plant sciences, University of Edinburg, UK, these cultured CMCs on the petri dish have the same characters as cells in the plant cambium. Theses results were published on NATURE BIOTECHNOLOGY, the NO.1 academic journal in biotech field, on NOVENBER, 2010.

9 JOURNAL:Nature Biotechnology (rank NO.1 in biotech field) TITLE: Cultured cambial meristematic cells as a source of plant natural products Nature Biotechnology 28, (2010) A REAL and SIGNIFICANT research achievement

10 9 CMC and DDC are ENTIRELY different cell types CMCs and DDCs have different ORIGIN. Cambial meristematic cells (CMCs,plant stem cells) - From plant cambium - Undifferentiated cells Callus cells (DDC, dedifferentiated cells) - From plant wound - Transformed from aged cells by dedifferentiation process Source: Nature Biotechnology 28, (2010)

11 10 CMC and DDC are ENTIRELY different cell types CMCs and DDCs are different MORPHOLOGICALLY Cambial meristematic cells (CMCs) - Cells are uniform - Cell growth is stable - many small vacuoles in the cell - No ageing Wild ginseng CMCs Ginkgo CMCs Tomato CMCs Wild ginseng DDCs Ginkgo DDCs Tomato DDCs Callus cells (DDCs, differentiated cells) - Cells are not uniform CMCs, o month CMCs, 1 month CMCs, 22 months - Cell growth is not stable - Only one big vacuole in the cell - Continually ageing (become brown color) Source: Nature Biotechnology 28, (2010) DDCs, o month DDCs, 1 month DDCs, 22 months supplementary

12 Cell dry weight (g/l) 11 CMC and DDC are ENTIRELY different cell types CMCs and DDCs have different GROWTH RATE. Cambial meristematic cells (CMC) - Single cell and small clumped cells suspended - keep growing Source: PSCI Callus cells (DDCs, differentiated cells) - Big clumped cells formation - The growth rate is decreasing, and stop eventually Needle DDC Embryo DDC Culture duration (days) Source: Nature Biotechnology 28, (2010) Supplementary

13 Ginsenoside (mg/kg FW) Abietanes (mg/kg FW) 12 CMC and DDC are ENTIRELY different cell types CMCs and DDCs have different PHYTOCHEMICALS YIELD Cambial meristematic cells (CMC) - Produce more phytochemicals - Synthesize more kinds of phytochemicals Yew Ddobyul CMC Taxamairin C Taxamairin A needle Yew DDC callus Callus cells (DDCs, differentiated cells) - Produce less phytochemicals - Synthesize less kinds of phytochemicals ginsenoside Rb1 ginsenoside F Wild Ddobyul ginseng CMC Ginseng stem callus DDC Source: Nature Biotechnology 28, (2010)

14 Gary Loake, expert of disease resistance mechanism of plant, co-author of Nature Biotechnology article This technology never successes before, and it will be a significant milestone in plant biotech. Robert C Gallo, Professor of University of Maryland, USA, co-finder of HIV This technology could overcome the limitation of plant natural products, and lead them to scientific research. Yung-Chi Cheng, Professor of Yale University, USA, called as the father of Chinese medicine This technology could isolate and culture plant stem cells from plants which are very important in traditional Chinese medicine, and it is very important for traditional Chinese medicine scientization and globalization. Namin Hyung, Professor of Sang Myung University, Korea, expert of plant tissue culture This team break the common consensus It is impossible to let every single cell express foreign protein, and this is a breakthrough for protein drugs industries. Susan C Roberts, Director of institute for cellular engineering, University of Massachusetts, expert of callus technology She ever said in the column News and views of the journal NATURE BIOTECHNOLOGY, Cambial meristematic cells are undifferented cells innately, and they have many different characters with dedifferentiated cells, which are used in the traditional plant tissue culture. CMC technology would be a more possible and economical method for phytochemicals production.

15 14 CMC platform could increase the number of rare plants CMC platform More than 50 year-old Korean wild ginseng Less than 20 plants per year Cambial meristematic cells isolation and culture technology platform (CMC platform) could clone and cultivate rare plants, to preserve their species and expand their value.

16 Amount 人森皂苷含量 of ginsenosides (mg/g) (mg/g) 15 CMC platform could increase the yield of phytochemicals Plant Plant CMCs Plants survived from years of severe environmental conditions have higher or more valuable phytochemicals. Such as 100 year-old ginseng, 1000 year-old yew tree CMCs would increase phytochemicals production dramatically after hours of elicited stress. Over the result of centuries of naturally growth General Ginsenosides 0.5 Rare Ginsenosides 0 General Ginsenosides 150 Rare Ginsenosides 50 year-old wild ginseng Wild ginseng CMC This is because that plant CMCs are immortal, high stress-resistance, and have the whole genome of plant. The elicited stress will activate the gene cluster involved in phytochemicals synthesis in the CMC.

17 16 CMC platform as a efficient protein-based drugs manufacturing system foreign gene CMC transfection Y Y Y Y CMC processing Y Y Y Y Y protein-based drugs antibody Vaccine hormone CMC Green fluorescent proteinexpressing CMC Source:PSCI Comparing with general protein manufacturing system (gene-modified animal milk, CHO mouse cell line, microbes, gene-modified plants, etc.), CMC platform has advantages in: Higher transgenic rate Lower cost Shorter duration No animal-to-human disease Lower allergenic Higher protein yield

18 17 Advantages of CMC platform Save money and space CMCs cultured in a 250L bioreactor for 1 month would produce more total rare gensinosides than many hectare of 6-yearold farming ginsengs. Key ingredients standardization The content of each batch of CMC cultures are 99.98% similar. It is very important for the industrialization of natural products. Environment free CMCs are cultured in a close system, which are particle free, temperature-controlled and moisture-controlled. Cells are influenced by environment factors no more. Rare ingredient Maximization We could optimize the culture condition to increase the yield of specific phytochemical. Much more effective than years of natural growth Pollution Free Manufacturing in a cgmp-grade factory and procedures. It prevents CMCs from microbes, heavy metals, pesticides, antibiotics, etc. Broad applications It could be used to foods, cosmetics, skin care products, health foods, medicines, protein drugs, species preservation, etc.

19 18 CMC platform is a competitive and exclusive patented technology worldwide 56 registered patents 31 patents pending Korea:15 Overseas:41 Korea:5 Overseas:26 Awards for achievements 2007, ISO9001 certification 2008, INNOBIZ certification 2011, New excellent technology (NET) 2011, Green Certification 2011, Korea Technology award, minister of knowledge economy award 2013, Grand Prize for small entity IP management Best practice award 2014, Award of Industrial Technology of Month Patent are registered in over 10 major countries, and covered the aspects of materials, methods and usages. These patents protect our core technology and ensure our advantages.

20 19 CMC platform could be benefit for many industries Pharmaceutical companies Food / Health food Cosmetics / Skin care Scientific research New Natural drug protein-based drugs raw materials for medicine raw material supply New raw material New ingredient New raw material New ingredient Species preservation Genetic improvement Academic research And YOUR BUSINESS

21 What we want you know Cambial meristematic cells isolation and culture technology platform Could open the whole genome of plant And release its potential in many aspects It is a revolutionary breakthrough in technology And has unlimited applications We want to cooperate with you And help you succeed ASAP We Culture The Future For You