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Insights in Enzyme Research
ISSN: 2573-4466
E u r o S c i C o n C o n g r e s s o n
Enzymology and
Molecular Biology
A u g u s t 1 3 - 1 4 , 2 0 1 8
P a r i s , F r a n c e
Enzymology 2018
The major component of extracellular matrix is the collagen and
collagenase enzymes are used to extract cells from biological tissues,
which the challenging goal is to obtain a high number of healthy and living
cells. The current collagenases utilized for regenerative medicine and cell
therapy are extracted from the culture of Clostridium Histolyticum and the
subsequent purification thereof of the bacterial proteins produced. The result
of this process leads to a blend containing different percentage ratios of
two main collagenase isoforms (class I and class II) plus a number of other
lytic enzymes (clostripain, trypsin like, caseinase activity, etc.). Such blends
present many limitations in terms of lot-to-lot consistency, variable enzymatic
activity and purity. In order to better control the extraction processes and
the enzymes formulation, we have generated recombinant collagenases of
class-I (COL G) and class-II (COL H), which allows efficient, customized and
standardized cell extraction procedures. These recombinant enzymes were
used together with thermolysin (as a generic proteolytic enzyme) in the
extraction processes of different cell types, for which the quantities of the two
classes of collagenases plus the neutral protease were precisely defined. The
current extraction procedure, with collagenases from C. histolyticum , is based
on a formulation that use a weight collagenase ratio, due to the impossibility
in determines the exact enzymes activity for each class of collagenases. Our
study performed with COL G and COL H to extract Langerhans islets from rat
pancreas highlighted how this formulation lead to variable results, while the
formulation based on the enzymatic activity ratio (COL G : COL H, determined
with the Grassmann method) allows a standardized and reproducible cell
extraction. Based on this results, several extraction protocols have been
improved, such as: cardiomyocytes from rat heart, chondrocytes from nose
or cow's hoof cartilage, hepatocytes from rat liver, osteoblasts from rat skull
cap and mesenchymal stem cells from rat adipose tissue. Each protocol was
optimized, using as parameters the phenotype and the number of extracted
cells, but also performing functional and /or differentiation assays.
Biography
Giulio Ghersi is Professor in Biochemistry and Applied
Biochemistry in Biotechnology, Element of Biochemistry and
Cellular Biology in Medical Engineering. Vice-Director Advanced
Technology Network Center (ATeN Center) University of
Palermo.CEO of ABIEL s.r.l. ) a spin-
off of the University of Palermo and of the Council National of
Research (C.N.R.) of Mediterranean Center for Human
Health Advanced Biotechnology (PONa3_00273 23 M€)PI
of “SIB: Advanced solutions using biomaterial by composite
matrix in repair and regeneration of articular cartilage using
non invasive techniques (PON01_01287 1,6 M€).Unit ABIEL PI
for Horizon 2020 project “Diabetes Reversing Implants with
enhanced Viability and long-term Efficiency – DRIVE” (0,9
M€).The research activities of greatest interest are currently
directed to the optimization of the extraction processes, of
cells for applications in the field of regenerative medicine and
tissue engineering, through the use of specific proteolytic
enzymes. As well as their use in nanostructured systems for
greater penetration into solid tumor masses, and the controlled
release of drugs and / or biomolecules with antitumor activity.-
Salamone M. et al (2016) “Proteolytic enzymes clustered in
specialized plasma-membrane domains drive endothelia cell
migration”. PLOS ONE, vol.11. Dispenza C. et al.(2012) Minimal
in Radiation Synthesis of Biomedical Functional Nanogels.
Biomacromolecules, vol 13, p.1805-1817.
Recombinant class-I and class-II collagenases: new
formulations in cells extraction
Giulio Ghersi
University of Palermo, Italy
Giulio Ghersi, Insights Enzyme Res 2018, Volume 2
DOI: 10.21767/2573-4466-C1-002