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DIY culture medium manual for DMEM and L15

Preparation manual for DIY DMEM and L15 (basal media) for animal cell culture experiments in kitchens - (Translation from original manual in Japanese)
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DIY culture medium manual for DMEM and L15

  1. 1. DIY culture media for kitchen experiments
  2. 2. Background 1: food safe DIY culture In past experiments, “Green Dakara” sports drink could be blended with DMEM up to 60% without losing cell culture efficacy. https://www.slideshare.net/2co/shojinmeat-project-cellular-agriculture-initiative-nov2018 However, all ingredients must be replaced with commonly available materials to enable kitchens DIY cell culture experiments. ※Research grade reagents = Not approved as food and not commonly available. Food or food additives that can be ingested (within limits) are commonly available. Basal media (DMEM, L15) was prepared entirely from commonly available materials and a DIY cell culture was demonstrated.
  3. 3. Background 2: composition of DMEM and L15 The biggest difference between DMEM and L15 is in the minerals. Other ingredients were assumed common. Precisely, DMEM and L15 have different amino acid compositions and DMEM uses glucose while L15 uses galactose DMEM⇒https://www.sigmaaldrich.com/life-science/cell-culture/learning-center/media-formulations/dme.html L15⇒ https://www.sigmaaldrich.com/life-science/cell-culture/learning-center/media-formulations/l-15.html DMEM L15 20 amino acids 20 amino acids Water- soluble vitamin B’s Water- soluble vitamin B’s Glucose (dextrose) Glucose (dextrose) CaCl2, MgSO4, KCl, NaCl, NaHCO3, NaH2PO4, Iron nitrate CaCl2, MgCl2, MgSO4, KCl, NaCl, KH2PO4, Na2HPO4
  4. 4. Ingredient 1 (Sugar, animo acids, vitamin B) 0.32g per tablet 0.22g amino acid (+sugar & fiber) 1 tablet (0.2g, incl. shellac shell) for 2L. Glucose for baking - Use 4.5g/L https://item.rakuten.co.jp/fancl-shop/5272/?i asid=07rpp_10095___e4-jpeuuk00-aev-615 07def-77bf-41df-8809-18631a826757 https://item.rakuten.co.jp/ kenkocom/x252760h/ https://item.rakuten.co.jp/a uc-garlic/budoutou-3000/
  5. 5. Ingredient 2 (Minerals for DMEM) https://item.rakuten.co.jp/m amoru/4571474270191/ https://item.rakuten.co.jp/m atsubayakuhin/10000000/ CaCl2 (calcium chloride) NaCl(salt) https://item.rakuten.co.jp/ jyugo/t-4987306019126/ Half Salt NaCl/KCl mixed at 1:1 MgSO4  (Magnesium sulfate) Mixing at 1:1 molar quantity makes NaH2PO4 ※Iron nitrate not included ・Only miniscule amount is needed. ・Iron nitrate is not commonly available - not a food additive. ・Replaceable with iron (III) chloride? baking soda https://www.amazon.co.jp/%E 3%82%AA%E3%83%AB%E3 %83%88%E3%83%AA%E3% 83%B3%E9%85%B8-%E3%8 3%AA%E3%83%B3%E9%85 %B8%EF%BC%98%EF%BC %95%EF%BC%85%E3%80% 8C%E9%A3%9F%E5%93%81 %E6%B7%BB%E5%8A%A0% E7%89%A9%E3%80%8D%E F%BC%95%EF%BC%90%EF %BC%90%EF%BD%87/dp/B0 062HL4PU phosphoric acid
  6. 6. Ingredient 2 (Minerals for L15) https://item.rakuten.co.jp/ mamoru/4571474270177/ MgCl2  (magnesium chloride) https://item.rakuten.co.jp/ kurimoto/100001011/ NaCl(salt) https://item.rakuten.co.jp/ jyugo/t-4987306019126/ Half salt 1:1 NaCl/KCl mixture https://www.amazon.co.jp/%E 3%82%AA%E3%83%AB%E3 %83%88%E3%83%AA%E3% 83%B3%E9%85%B8-%E3%8 3%AA%E3%83%B3%E9%85 %B8%EF%BC%98%EF%BC %95%EF%BC%85%E3%80% 8C%E9%A3%9F%E5%93%81 %E6%B7%BB%E5%8A%A0% E7%89%A9%E3%80%8D%E F%BC%95%EF%BC%90%EF %BC%90%EF%BD%87/dp/B0 062HL4PU Mixing at 1:1 molar quantity makes NaH2PO4 https://item.rakuten.co.jp/m amoru/4571474270191/ MgSO4  (Magnesium sulfate) potassium carbonate Mixing at 1:1 molar quantity makes KH2PO4 baking soda phosphoric acid
  7. 7. 1X Solution 20X Solution Synthesis method DMEM: Table salt 0.58g baking soda 0.37g Half salt 0.13g L15: Table NaCl 0.95g Half salt 0.10g 125ml water 1 amino acid tablet + 99ml water 1 vitamin B pill General strategy for preparation ・Prepare 1X concentrated solution for salts, glucose, and amino acid, for which large quantity is needed. ・Prepare 20X solution for vitamin B’s and other minerals, for which only trace amount is needed. ・Prepare phosphates from phosphoric acid and mineral salts. DMEM: CaCl2 0.50g MgSO4 0.25g baking soda 0.19g Phosphoric acid diluted 10X 1.4ml L15: CaCl2 0.28g MgSO4 0.20g MgCl2 0.19g K2CO3 0.06g Phosphoric acid diluted 10X 1.4ml Mix 1X solution and 20x solution by 19:1, add glucose at 4.5g/L and bon apetit
  8. 8. Scenes from the experiment ・Filtration is needed, as the tablet excipients need to be removed. ・Wait until the suspension settles to avoid clogging the filter paper. Holding position with a peeler Filtered 1X solution
  9. 9. Cell culture demonstration Primary culture of chicken foetal heart cells by DIY-DMEM: Successful cell adhesion and proliferation (culturing experiment by @thesow41 on twitter) https://www.nicovideo.jp/watch/sm33104702 ※DIY-L15 to be tested
  10. 10. Participate in improving this manual! https://docs.google.com/presentation/d/1cNedlmZcW7JOKB-gM itL3aIix1YXd5gi0hhlYz1mVqQ/edit#slide=id.g5683e89615_0_0 Source file openly accessible and editable