213The results of this study also confi rmed the proposition made by Tommaso and
co-wokers ( 2012 ) that ASCs are sturdier cells than adipocytes and can withstand
centrifugal forces of up to 3000 rpm (Condé-Green et al. 2010 ). The oil fl oating
material layer seen in centrifuged samples was previously analyzed by Novaes and
co-workers ( 1998 ). They used gas chromatography to examine the nature of this
fl oating oil material and identifi ed the substances as lauric acid, stearic acid, pal-
mitic acid, and araquidic acid, where the highest volume was occupied by palmitic
acid (Novaes et al. 1998 ) indicating contamination, which supports the practice of
removal of the oily supernatant.
Various aspects surrounding the centrifugation process during the isolation pro-
cedure can infl uence the isolation yield. Baschert and co-workers suggested that
centrifugation forces greater than 100 g are not appropriate for autologous fat trans-
plantation as they observed an increased quantity of oil possibly due to adipocyte
destruction (Baschert et al. 2002 ). In contrast, Kurita and colleagues found that
more than a 100 g centrifugal force could be used for autologous fat grafting, since
the increased oil portion does not necessarily mean an increase in adipocyte destruc-
tion, but rather an increase in the separation of oil from the adipose portion (Kurita
et al. 2008 ). Centrifugation of adipose tissue separates fat cells from lipid, blood
cells, water, and water-soluble ingredients such as proteases and lipases, but does
not shift ASCs between the adipose and fl uid portions, possibly due to the strong
adherence to adipose tissue or since they are resident within the adipose tissue. It
was also shown that increased centrifugal forces compacted the adipose portion
more and therefore concentrated the red blood cells within the adipose portion
rather than shifting the red blood cells into the fl uid portion. In contrast to mature
adipocytes, it was found that the yield of ASCs in culture for 1 week was consistent
up to 3000 g but decreased with centrifugal forces of more than 3000 g (Kurita et al.
2008 ). Dickens and co-workers demonstrated that gentle centrifugation produced
the highest cell viability, whereas long periods of centrifugation resulted in the
selection of the most proliferative ASC subpopulation (Dickens et al. 2009 ).
Another factor to consider in the isolation process is the effect of seeding density
on cell proliferation. Fossett and colleagues ( 2012 ) showed that low seeding densi-
ties increase the proliferation capacity in vitro. The effect of seeding density on
MSC proliferation was demonstrated with bone marrow-derived MSCs that were
seeded at 100 cells/cm^2 and reached their target of 200 × 10^6 cells 4.1 days faster
than cells seeded at 5000 cells/cm^2 (Both et al. 2007 ). Similar results were observed
by Lode and co-workers in 2008 using synovial fat pad MSCs seeded on three-
dimensional scaffolds (Lode et al. 2008 ). Witzeneder et al. applied different ASC
seeding densities for expansion (3200 cells/cm^2 ) and lineage induction experiments
(7000 cells/cm^2 ), while Lindroos et al. seeded cells at 5000 cells/cm^2 for ASC
expansion purposes. Krähenbühl et al. found good cellular expansion with seeding
densities of 325, 750, 1500, and 3000 cells/cm^2 but in contrast to Fossett and col-
leagues found increasing yields with higher densities (Krähenbühl et al. 2015 ;
Lindroos et al. 2009 ; Witzeneder et al. 2013 ). Fink and co-workers found that ASC
expansion is optimal between 100 and 200 cells/cm^2 with a range of 50, 100, 200,
and 800 cells/cm^2 (Fink et al. 2011 ). The literature therefore does not provide
10 Harvesting and Collection of Adipose Tissue for the Isolation...