Tissue Culture Laminarflow Hood

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??????? ???????????? ???????????????????????????
???????????????????????
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????????????

• Tissue Culture (Laminar-flow) Hood
• CO2 incubator
• Aspiration pump
• Tissue culture substrate
• Media Supplements

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1. Tissue Culture Hood

• Laminar-flow Hood???? Laminar-flow Cabinet
• ??????????????????????????????????
• ????????????? High Efficiency Particulate Air
  (HEPA) filter

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???????? UV ??????????????
http//en.wikipedia.org/wiki/ImageUV-ontsmetting_
laminaire-vloeikast.JPG
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http//www.globalrph.com/images/verticalhood.jpg
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http//www.globalrph.com/images/horizontalhood.jpg
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Class II Biohazard Cabinet

• ???????????????????????????????????? ????
  ?????????????????? ???????????? ????????
• ????????????????????????????????
• ??????????????????????? (??????? human cells)

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2. CO2 Incubator

• ?????????????????????? CO2 ?????? 5
• ??????????????????????????????? ?????????? CO2
  ????????? ???? incubate ?? dry incubator ???? hot
  room ??????
• Petri dish ??? multi-well plate
  ???????????????????????????????

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• ????????????????????????????????????????????? CO2
  incubator
• ?????????????????????????????????????????
  ???????????????????????????????

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3. Aspiration Pump

• ?????????????????????????????????????????????????
• ???????????????? sterile pipette ????????
• ??????????????????????????????????? ????
  gluteraldehyde ???? hyperchlorite

Freshney, 1994
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Freshney, 2005
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4. ???????????????

• ??????????????????????????????????????????????????
  ???????????????????????????????
  ??????????????????????? ?????????????????????
• ??????????????????????????????????????????????????
  ??????????????????????????????????????????????????
  ?????????
• ??????????????????????????????????????? (adhesion)

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?????????

• ?????????????? ??????????????????????
• ??????????????????????????????????????????????????
  ??????????????
• ??????? ?????????? ???????
• ????????????????????????? ???? NaOH
  ?????????????????????????????????????????
  ????????? neutralize ???? acid wash

Freshney, 1994
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Disposable Plastics

• Polystyrene flasks ???????????????? ?????
  ?????????????????????????????????????????????????
  ???????(reproducible)
• Polystyrene ??????????? hydrophobic
  ????????????????????????????????
• g-irradiation ???? chemical treatment

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Disposable Plastics

• ?????????? ?????? ???????? (T-25, T-75, T-125)
• Sterile ?????? ????????????
• ??????????? detergent ????????????????????????????
  ????????????
• ????????????? ??????????????????
• ????????? polyvinylchloride (PVC),
  polycarbonate, melinex, thermanox (TPX) ???????

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Petri dish Multi-well Plates

• ?????????????????????? 35-mm, 100-mm, 6-well,
  12-well, 24-well, 96-well
• ??????? ?????????? media ????????? ???
  contamination ????

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(No Transcript)
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5. Media Supplements

• ?????????????????????????????????????????????????
  
• ?????????????????????? embryo extract, lymph,
  protein hydrolysate, etc.
• ?????????????????????????????????????????????????
  ?????????????????????????????????????
  (qualitative consistency)

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Chemically Defined Media

• Eagles BME, Eagles MEM
• Dulbeccos Modification of Eagles MEM (DMEM)
• CMRL 1066
• RPMI 1640
• Hams F10, Hams F12
• Etc.

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??????????????????? Media

• Essential amino acids (tryptophan, methionine,
  valine, threonine, phenylalanine, leucine,
  isoleucine, lysine) tyrosine cysteine
• Vitamins
• Salts
• Glucose
• Organic supplements pyruvate, citric acid cycle
  intermediates, proteins, peptides, nucleotides,
  nucleosides, lipids

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pH of the Media

• ?????????????????????????? pH 7.4
• Fibroblasts ????????????? pH 7.4-7.7
• ???????????????????????? pH 7.0-7.4
• Epidermal cells ??????????????? pH 5.5 ???
• Phenol Red ???????? pH 7.4, ???????? pH 7.0,
  ??????????? pH 6.5, ???????????????? pH ???????
  6.5, ?????????????? pH 7.6, ???????????? pH
  ??????? 7.8

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Buffering Capacity of Media

• ????????????????????????????????????? pH
  ????????????????????????
• ???????????????????????? ???? bicarbonate buffer
  ??? HEPES
• HEPES ???? buffer ??????????????????? pH 7.2-7.6
• ???????????????? ???? 20 mM HEPES, 10 mM
  bicarbonate

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Serum

• ??????????????????? growth factors, adhesion
  factors, ??? antitrypsin activity
• ???????????????????????? (undefined)
  ??????????????????? ???? minerals, lipids,
  hormones
• ??????????????????? fetal calf serum, bovine
  serum, horse serum, ??? human serum
• ???????????? (???? HeLa ??? L929) ??????????
  serum-free media

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Serum Substitutes

• ????? ?????????????????? serum ????????????????rep
  roducibility ??? ??????????????????????????
• SerXtend (NEN)
• Ventrex (Ventrex Laboratories, Ltd.)
• Nutricyte (Brooks Laboratories)
• Nu-serum (Collaborative Research)
• CLEX (Dextran Products, Ltd.)

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Other Supplements

• ?????????? (???? Penicillin, Streptomycin,
  Gentamycin sulphate, Amphotericin B)
• Embryo extract
• Conditioned medium

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???????? Media ?????????

• ????????? autoclave ??????????????????????????????
  ??????????????????????????
• ?????????????????? 0.22 mm membrane

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• ??????? ? ????

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  ?????????????, ?????????????, ???????????
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????????? ????????????????????
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  ???????? ??????? ??????????????????? ??????
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• ????????????????????? ??? ?????????
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  ????? ???????...

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