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DIY cell culture manual (& the roadmap to DIY cell-based meat)

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Instructions for DIY cell culture experiment in kitchens and the roadmap to "growing meat at home" involving tissue engineering - This manual is editable by everyone for improvements!

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DIY cell culture manual (& the roadmap to DIY cell-based meat)

  1. 1. DIY Cell-based meat Roadmap & manual 

  2. 2. Roadmap to DIY cell-based meat DIY cell- based meat Edible cell scaffold DIY cell culture protocols Consolidation of “DIY cell culture” Establishment of communication infrastructure & communities Control of differentiation and tissue formation Credits: @okgw_ ) Development of cell culture hardwares Cell culture device with pumps Implementation of high-efficiency cell culture hardware Development of tissue culture hardware Replace FBS or egg yolk to conditioned medium DIY serum alternatives Development of automated cell culture setups Development of growth factor production protocols based on coculture Efficient cell culture protocols Demonstration of “DIY tissue engineering”
  3. 3. Overall steps of cell culture Cell extraction P4 Cell centrifuge P5 Cell cyropreservation P13 Cell culture P10,11 Cell culture medium P6 Preparation of cell culture environment P8 Cell passage P12 Observation of cells P9 Other cells and prospects P14 Supplements preparation P7
  4. 4. Cell extraction Incubate fertilized chicken egg for 8~12 days and extract cells from foetal tissues Extraction from live tissues Live cells are needed for cell culture (the cells from meat and fish in supermarkets are most likely to be dead) Cryopreserved cells Foetal cell extraction Quickly defrost the cyrotube containing the frozen cells using 40℃ water (cells die if done too slowly) Purchased frozen cells or cells prepared by methods on P13 Mince raw tissue, incubate at 37℃ for 40 minutes after adding collagenase. Peel off cells from the cellular tissues. (These are the steps for cell dispersion.) It is possible to use papain (which can be purchased by commoners) instead, but you may need to adjust incubation time to 3-5 minutes as it is “strong” https://drive.google.co m/file/d/1dBDO9-4jvric 74R_3sPiiZyLpCbHZG bh/view http://zacmayu.hatenab log.com/entry/2018/03/ 04/235700  One of many cell retailers
  5. 5. Cell centrifuge Attach Falcon Tubes using duct tape. If the tube weighs 30g, the centrifugal force is 5~6kg. Fan = 800~1000rpm 20cm diameter: 143G 30cm diameter: 200G Centrifuge of cell mass for about 1-3 minutes at 150-200G The cells end up here
  6. 6. The basal medium https://www.slideshare.net/2co/diy-cult ure-medium-manual-for-dmem-and-l15 “Sports drink culture medium” 「DIY-DMEM & L15 Leibovitz」 https://www.slideshare.net/2co/shojinmeat-proj ect-open-source-cellular-agriculture-initiative A brand of sports drink with pH and osmotic pressures adjusted could be mixed up to 60% with DMEM without losing cell culture efficiency. Preparation of basal medium (DMEM and L15) solely from accessible ingredients (food and food additives) Mixing of sports drinks with DIY-DMEM and DIY-L15 media is a work in progress. Mix 1X solution and 20x solution by 19:1, add glucose at 4.5g/L and bon apetit
  7. 7. Growth factors (growth hormones) are needed to signal cells to proliferate Yeast extract + egg yolk 0.1%Serum, i.e. Foetal Bovine Serum (FBS) Co-culture / Conditioned media Conventional very expensive option used in university and corporate labs - not available in supermarkets, and has safety concerns if for food. One of the past experiments shows yeast extract containing growth factors, and missing ones can be sourced from egg yolk for 5 days. The effectiveness was comparable to FBS. ※More tests are needed to confirm https://www.nicovideo.jp/w atch/sm31104033 https://twitter.com/shoj inmeat_jp/status/1104 214121375920129 Literature suggest mutual stimulation of cell growth in the order of Placenta⇒Liver⇒ Muscle⇒Placenta (Ohlsson C et al., 2009, Endocr Rev. Francis GL, 2010, Cytotechnology) Fish cell growth can be induced by co- culturing with kidney cells. Medium supplements GF’s
  8. 8. Setting up a cell culture environment Hand-towel warmer hack DIY raspberry-pi incubator Reptile mat in a styrofoam box ・Altering the AC in voltage from 100V to 30V reduces the operating temperature from ~70℃ to ~38℃ ・Portable drink warmer/cooler boxes also do. If culturing fish cells, the room temperature (controlled by an air conditioner) is sufficient (Credits; @okgw_ )(@earthlyworld) ・”Sotacubator” ・~$70 ・On GitHub ・Open source https://github.com/sotakan/RPi-Incubator ・$20~$40 ・Beware of temperature gradient and condensation Drink boxes also work
  9. 9. Atmospheric and pH control CO2 forms by mixing baking soda with citric acid. at 3:1 molar ratio, 4:3 weight ratio. (25L CO2/mol) DMEM mediums need ~5% CO2 to buffer the pH. L-15 Leibovitz medium does not need it, but at a smaller buffer capability. This may be compensated by the quantity. Sparkling water Baking soda + citric acid A lot of culture medium By using a lot of medium, the pH and concentration may perhaps drift less with no atmospheric control. ※Not confirmed yet
  10. 10. Contamination countermeasures in cell culture Spraying 70% ethanol DIY Clean Bench Antifungal effect using egg white Experiments often fail from mold and bacterial contamination. Solutions: Don’t let mold pollens in, give the media “immunity” Pharmacies are the most likely place to find ethanol in most countries An air purifier combined with a box or large plastic storage box makes a DIY “clean bench”. Egg white contain lysozyme, a natural antimycotic. A mold-resistant medium can be prepared by mixing 5~10% of egg white.
  11. 11. Observation of Cells (microscopes) Smartphone Microscope Standard school microscope DIY Inverted microscope 40X is enough to observe cells. An inverted microscope is ideal as it can see cells stuck on the bottom of a cell culture petri dish. From ~$10. May feel difficulty focusing. From ~$200. It is not an inverted microscope - be careful not to let the objective lens touch the medium when focusing on the cultured cells. Cells on the bottom of the petri dish A DIY inverted microscope using a junk iPhone lens and a Raspberry Pi - may have web connection to post on Twitter. http://biohacker. jp/c/BH222.html
  12. 12. Cell passaging Detach cells from the dish Remove the medium and pour saline or PBS. The cells are bound on the dish and don’t easily get washed away. Add 100μl of PBS or 1mL saline + trypsin/EDTA solution and incubate for 1 minute at 37℃. Wash cells Pour out the red culture medium and wash the cells with colorless PBS/saline Passaging = Transferring of cells to a new dish and medium If using scaffold material... If using cellular scaffold, cut the scaffold into smaller pieces and place on new scaffold. There is no need to remove cells from the scaffold. Dried konjac scaffold saline+trypsin 37℃ 1 min cells come off by tapping the dish Transfer cells into another vessel Detach cells from dish by tapping the bottom of the dish. Transfer into a test tube, add 3ml medium and centrifuge. move to a test tube for centrifuge
  13. 13. Cell Cryopreservation Cool down to -70℃ using dry ice and ethanol or nail varnish (acetone). Quickly freeze the cells (with cryopreservation agent) in the cyrotube. After instant freezing, cell can be stored in a standard kitchen freezer. - Freezing cells before formation of ice crystals, cell rupture and death. Centrifuge cells Add cryopreservation agent Freeze quickly Centrifuge the mixture of trypsin, saline, cells - remove the fluid component leaving the cells at the tip⇒P6 Add 1~2ml cryopreservation agent to the centrifuge tube. Depending on the amount of cells obtained, cell mass can be split to multiple tubes. Cells pile up at the tip upon centrifuge Cryotube for cryo- preservat ion The most important thing is to protect cells from ice crystals If slowly frozen, the sharp ice crystals break the cell membranes causing cell death.
  14. 14. Varieties of cells & Future experiements - Replace collagenase with papain - Establish more stable DIY cell culture protocols - Mixing of sports drinks with DIY basal media - Further experiments with “0.05% egg yolk as FBS replacement” - Co-culture experiment to procure growth factors - Cell culture without 5% CO2 by using large quantity of DIY DMEM, Differences between species Differences between cell types The cell culture temperature may vary among species. The efficiency may vary from “zero”, “not ideal” to “sufficient” even if the temperature is not right. Different cells need different growth factors. Many types of cells need FBS, and liver cells also need HGF to proliferate. Replacements and conditioned medium (co-coculture) can be used to obtain growth factors, even for brain nerve cells or iPS cell culture, but this remains a future work. birds & mammals DMEM 38℃ fish DMEM ・L15 25℃ shellfish & insects L15 15~25℃ Future experiments
  15. 15. Participate in improving this manual! https://docs.google.com/presentation/d/1cNedlmZcW7JOKB-gM itL3aIix1YXd5gi0hhlYz1mVqQ/edit#slide=id.g7d2898c787_0_9 Source file openly accessible and editable

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