학회: 29th AACS - continuouns stem cell expansion without trypsin which changes cell characters |
Continuous stem cell expansion without
trypsin which change cell characters.
Heeyoung Lee. M.D., Soyoung Jang, M.B., Hyunjin
Yang. M.D.,
Baroyl & Kangnam Plastic Surgery.,
Seoul, Korea.
Expansion of cell number would be the most
important thing for the success of human cell therapy. During this cell
expansion, trypsin dissolution is still necessary to move cells to new attachment
surfaces. However, this trypsin is regarded as a strong irritating chemical and
mutagen to cells, and it is the only variable to decide the number of passages.
Theoretically, many articles reported that the characters of cells might not be
changed under around P12. On the other hand, it could mean that the cells over
P12 might not be safe to use clinically. Always this passage number is the
major restriction of cell number expansions in clinical uses for cell therapy.
We have been tried to overcome this
'trypsin restriction'. As methods of cell detachment without trypsin, thermo sensitive
sol-gel polymer coating on scaffolds, mechanical forces like water sharing
movement, friction, scraping could be useful. We were partially successful in ‘trypsinless’
micro beads scaffolds cell expansion using with intermittent stirring and
gradual increase of the scaffolds, however we still could not find any steady standardized
protocol and the method of monitoring for so variable level of activities of
cells from different individuals with different ages.
Film overlapping and mechanical scraping
would be good for trypsinless cell expansion with easy monitoring. Material and
shape of these films are variable. We have been tried 50 um thick polyethylene
film as a roll or simple square overlapped films in DMEM with FBS. We expanded
the ASCs number 32 times within 3 weeks without trypsin treatment. For the
movement of cells to new surfaces, we just used method of sequential film
overlapping with increasing the area of cell attachment surfaces. When we need
the cells for therapy, we scraped cells mechanically and use directly to
patients. However, for the uses of I.V. we needed to use trypsin EDTA for each
cell separation.
As a result, when we use 200% overlapping, we
diminished the trypsin uses under 1 ~ 2 from cell sources to 20 times expansion
of cell numbers. Total time consuming was also decreased to 60% comparing with
usual dish cultures with same cells. So, we could find several reasons to
believe that unlimited cell expansion without trypsin toxicity with
intermittent harvesting could be possible. If we overlapped the film over 400%
or more, we could develop to save the time and effort more, standing away from
the controversy of trypsin toxicities.
As a conclusion, mechanical cell detachment
and serial attach surfaces can minimize trypsin uses in continuous harvest and
stromal cell culture.
Continuous film overlapping cell expansion(FOCE)
without trypsin toxicities.
Heeyoung Lee. M.D., Soyoung Jang. M.B.,
Hyunjin Yang. M.D.,
Baroyl & Kangnam Plastic Surgery., Medikan
Co, Seoul, Korea
Objects;
Continuous stem cell expansion without
trypsin not to change cell characters.
Material & methods;
Expansion of cell number would be the most
important thing for the success of human cell therapy. During this cell
expansion, trypsin dissolution is still necessary to move cells to new attachment
surfaces. However, this trypsin is regarded as a strong irritating chemical and
mutagen to cells, and it is the only variable to decide the number of passages
We have been tried to overcome this
'trypsin restriction'. Film overlapping and mechanical scraping would be good
for trypsinless cell expansion with easy monitoring. Material and shape of
these films are variable. We have been tried 50 um thick polyethylene film as a
roll or simple square overlapped films in DMEM with FBS. For the movement of
cells to new surfaces, we just used method of sequential film overlapping with increasing
the area of cell attachment surfaces. When we need the cells for I.V. therapy,
we scraped cells mechanically and used water sharing filters directly to
patients.
Results;
We expanded the ASCs number 32 times within
3 weeks without trypsin treatment. When we use 1 : 2 overlapping, we didn't use
the trypsin uses to 20 times expansion of cell numbers. Total time consuming
was also decreased 50% comparing with usual dish cultures with same cells. So,
we could find several reasons to believe unlimited cell expansion without
trypsin toxicity. If we overlapped the film 1 :4 or more, we could save the
time and effort more.
We have been treated 15 patients with I.V.
for general ant-aging, 26 patients for local injection for wound healings.
Conclusion;
Mechanical cell detachment and serial
attach to new surfaces(Film Overlapping Cell Expansion; FOCE) can get rid of trypsin
toxicities in continuous harvest and stem cell culture for almost uses in
clinical situation.