Aseptic Techniques

1
???????????????????????????????/???????????????
2
????????????

• ??????????????? (Aseptic Techniques)
• ??????????? Subculture
• ?????????????????? (Single-cell Cloning)
• ???????????????? (Colony Picking)
• Fluorescence-activated Cell Sorting (FACS)
• ?????????????????????? Magnetic Beads
• ??????????????????????????????? 2 ????
• ???????????????????? (Cell Fusion)
• ?????? Cell Synchronization

3
1. ??????????????? (Aseptic Technique)

• ??????????????????????????????????????????????????
  ?
• ?????????????????????/??????????????
• ??????????????????????????????
• ?????????????????????????????????????
• ???????? culture ??????
• ?????????????????????????

4
??????????????? (???)

• ??????????????????????
• ????????????????????????
• ????????????? (antibiotics) ?????????????????
• ???????????????????????????????? (UV, 70
  alcohol)
• ???????????????????????????? ?????????????????????
  ??????????????????????????????????????

5
?????????????????? (contamination)

• ???????????????????????????????????????????????
• ??????????????????????????????????????????????????
  ?????????????
• ??????????????????????????????????????????????????
  ????????????? biohazard material
  ?????????????????????????????

6
2. Subculture Technique

• ????????????????????/???????????????????????????
  
• ????????????????????????????
• ?????????????????????
• ???????????????????????????????????
• ?????????????????????????????????????????????
• ??????????????????????????????????????????????????
  ???????????????? (????????????? subculture)

7
???????????? subculture

• ???????????????????????????????????
  ????????????????????????
• Dilute ??????????????????????
• ????????????? (??? hemocytometer ?????????????)
• ???????????????????????????????? (???? 1
  ???????????? T-75 flask) ????????????????????????
• ???? fresh medium

8
??????????????????????????????????????????????????
??

• ????? adhesion molecules ?????????????????????
  trypsin
• ?????????????????????? adhesion molecules
  ????????? chelating agents ???? EDTA
• ??????????????????????????????????????????????????
  ??

9
????????????????? Hemocytometer
The volume of one quadrant is known to be 0.1 ml
10
????????????????????????????????????????
11
????????????????? Coulter Counter
Readout
Amp
The counter detects change in electrical
conductance of a small aperture as fluid
containing cells is drawn through. The cell, a
non-conducting particle, alters the effective
cross-section of the conductive channel.
http//www.wps.com/about-WPS/WPS/coulter-counter.J
PG
12
3. ?????????????????? (Single-cell Cloning)

• ??????? limiting dilution
• 5 cells/ml
• ??????????? 96-well plate
• 0.1 ml/well
• ????????????????????????
• ?????????? well ???????????????

X
X
X
X
96-well Plate
13
4. ???????????????? (Colony Picking)
????
trypsin
Cloning Rings
???????????????????????? pipette tip
14
5. Fluorescence-Activated Cell Sorting (FACS)
????????????? Fluorochrome-labeled monoclonal
antibodies
http//www.bio.davidson.edu/courses/genomics/metho
d/FACS.gif
15
http//www.gsf.de/Forschung/Institute/zs/common/pr
inciple123.gif
16
?????? FACS ???????????????????????????????
??????? 1
??????? 2
??? X ??????????? fluorescence1 ??? Y
??????????? fluorescence2
??? X ??????????? fluorescence ??? Y
??????????
17
6. ?????????????????????? Magnetic Beads
????????????????antibody ???magnetic beads
????????
18
7. ??????????????????????????????? 2 ????

• ????????????????????????? 2 ???????????
  (co-culture technique) ???????????????????????????
  ?????
• ??????????????????????????????????????????????????
  ?? (contact-dependent interaction)
• ?????????????????????????????????????????????
  (paracrine interaction)

?????????????????????? 2 ????????????????????????
??????????????
19
??????????????????? 2 ???
Paracrine signals are released by cells into the
extracellular medium in their neighborhood and
act locally.
Contact-dependent signaling requires cells to be
in direct membrane-to-membrane contact with each
other.
20
??????????????????????????????? 2 ???? ??????
Transwell Co-culturing System

• ??????????????????????????????????????????????????
  ???????? contact-dependent ???? paracrine

http//www.corning.com/lifesciences/cell_culture/i
mages/transwell20diag.gif
21
???????? Transwell Culture Systems
http//www.drzeydanli.com.tr/docs/cellculture/tran
swell/transwell.html
22
8. ?????? Cell Fusion

• Cell fusion ????????????????????? 2 ?????
  ?????????????????? ???????????????????
  heterokaryon
• ??????????? heterokaryon ?????????????????????????
  ??????????????????????????????? cell fusion
• Cell fusion ??????????????????????????????????????
  ??????????????????????????????????????????????????
  ?????

23
???????????? cell fusion ?????????????????????????
?????????????????????????????????????????????????
?????????????????
Campbell and Reece, 2002
24
Cell Fusion (???)

• ??????????????????????????????????????????????????
  ???????
• Heterokaryons ?????????????????????????????
  (???????????????????????????)
• Mouse-human hybrid cells ?????????????????????????
  ?????????
• ???????? hybrid cells ??????????????????????????
  11 ??????????????????????? insulin ????????????

????????????????????????????????????????????????
11
25
????? Cell Fusion

• ??? inactivated virus ???? ??????? polyethylene
  glycol
• ??????????????????????? cell membranes ??? fuse
  ?????????? (????? membrane fluidity)
• ???????? fused cells ?????????????????? selective
  medium

http//biology.kenyon.edu/slonc/bio3/2000projects/
marshall_g/my_cell_fusion.JPG
26
Hybridoma

• ????????????????????????? monoclonal antibody
• ???? cell fusion ??????? B lymphocyte ???
  immortal B lymphocyte tumor (myeloma)
• ????? hybridomas ????????????????????? 2 ?????
  ????????????????????????????????????????????
• Selective medium ????????? HAT medium
  (hypoxhantine, aminopterin and thymidine)

27
Hybrid cells are recovered through their ability
to survive in HAT (hypoxhantine, aminopterin and
thymidine so-called Littlefield's medium
Science 145 709, 1964) whereas the parent
myelomas die in this medium because of mutation
at the Tk1 (thymidine kinase 1, chromosome 11) or
Hprt (hypoxhantine guanine phosphoribosyl
transferase, X chromosome) loci.
http//nfs.unipv.it/nfs/minf/dispense/immunology/s
omcellfus2.jpeg
28
9. ?????? Cell Synchronization

• ????????????????????????????????????? cell cycel
• ???? mitotic shake-off
• ???? blockade (?????? S-phase inhibition ???
  Nutritional deprivation)
• ????????????????????? cell cycle progression
  ???????? karyotype

29
Mitotic Shake-off

• ???????????????????????????????? mitosis
  ??????????????????????????????????????????????????
  ??????
• ??????????????????????????????????? (shaking off)
  ?????????????????????????????
• ????????????? CHO cells ?????? sublines ??? HeLa
  cells
• ??? incubate ???????? 4 oC, 0.5-1 ???????,
  ???????????????????? 37 oC ???????? 2-3 ???????
  ??????????? yield

30
DNA systhesis inhibition

• ???? cell cycle ?????? S-phase
• ??? Thymidine, hydroxyurea, cytosine arabinoside,
  aminopterin, etc.
• ???????????????????????????????????
  ????????????????????? block

31
http//www.med.unibs.it/marchesi/cellcycle.gif
32
Nutritional Deprivation

• ???? cell cycle ?????? G0-phase
• ???????????????????????????????????????????????
  serum ???? isoleucine ???????? 24 ???????
• ???????????????????????? ???????????? cell cycle
  ????????
• ??????? (gt80 synchrony) ??????????
• ???????? 2 ????????? 60 synchrony
• ???????? 3 ????????