18 Dic How to get ADSC and SVF, ideal for routine clinical use, from adipose tissue
Several studies demonstrate that ADSCs
have the capability to differentiate
into different cell lineages
(such as adipocytes, osteoblasts,
cartilaginous cells, hepatocytes
and myocytes (5-7).
The gold standard to obtain ADSC and SVF is enzymatic digestion, which allows to obtain a large number of cells rapidly. However, the European legislation consider enzymatic digestion as a substantial manipulation and therefore not feasible in the clinical practice. (8).
How to easily get high grade ADSCs and
SFV in a very fast and standard way
which are ideal for routine clinical use?
Micro-grafts tissue isolation with RIGENERA-HBW technology
Rigenera consists of a medical device (Class I), called Rigeneracons and a rotor that allow the device to rotate at 80rpm. The device mechanically selects progenitor cells, that are smaller than 70 microns, thanks to a grid.
In his study, De Francesco (note A) compared the biological property of ADSCs obtained with with enzymatic digestion and mechanical fragmentation with Rigenera-hbw technology, analysing:
- viability and number of cells (Trypan blue) at 0, 72h and 10 days
- expression of mesenchymal markers (citofluorimetry)
- growth in culture
- expression of genes involved in differentiation and staminality (REAL-TIME PCR)
Adipose tissue harvesting was performed on four women subjected to liposuction for aesthetic purposes, with ages ranging between 28–50 years, (according to the ethical guidelines set by the review board for human studies of AOU “Ospedali Riuniti”, Ancona, Italy) using the BEULI protocol, with water-jet and 38mm cannulas. The obtained tissue was washed twice with sterile saline solution and prepared for enzymatic or for mechanic digestion.
For Rigenera fragmentation : 12 ml of lipo-aspirate were inserted in the Rigeneracons with 4ml of culture medium. Fragmentation was performed for 30 and 45 s, in order to determine which of the two timings was most suitable to obtain a better vascular stromal fraction, without affecting its vitality.
For enzymatic digestion: the remaining part was digested with the standard protocol for collagenasi, then centrifuged and filtered on a 70-micron filter. Both suspensions were cultured for the subsequent analysis.
Results of this study demonstrate that:
in the suspension obtained processing
12 ml of lipoaspirate with Rigenera there
are about 4200 viable cells,
with elongate shape,
intact membrane and nucleus.
In some cells, small lipid droplets are
also detectable. The number of cells,
if compared with the enzymatic digestion,
is lower, but viability is high
at every timing (0, 72h and 10 days),
and also the proliferation
rate is great.
Citofluorimetrical analysis demonstrates that cells obtained with RIGENERA have:
- high positivity at mesenchymal stromal markers CD105, CD90, CD73, CD117), as previously described (9)
- negativity for hematopoietic markers (CD31, CD 45)
In order to confirm the potential as a stem cell of isolated cells the mRNA of CD73, CD105, CD90, CD45 and CD34 were extracted and measured by real-time PCR, enzymatic fat digestion was used as control. A significantly comparable mRNA expression for all the stem cells’ markers was detected in the Rigenera method compared to enzymatic digestion. No differences between 30 and 45 seconds were detected.
In conclusion, enzymatic digestion
is currently the gold standard procedure,
providing more adipose-derived
stromal cells and SVF from adipose
tissue, while mechanical
fragmentation with Rigenera
obtains cells aggregation with characteristic
and quality ideal for routine clinical use.
Moreover, this protocol is simple, rapid
and reproducible, providing
cell–enriched stromal vascular fraction
(Note A) This documents is based on the following paper : De Francesco et al A Non-Enzymatic Method to Obtain a Fat Tissue Derivative Highly Enriched in Adipose Stem Cells (ASCs) from Human Lipoaspirates: Preliminary Results. 2018 Int. J. Mol. Sci. 2018, 19, 2061; doi:10.3390/ijms19072061
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