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Tag-less and gentle isolation, selection, and sorting of stem cells
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Celector®
Sorting your cells as easy as it gets
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Abstract
Stem cells are distributed in all tissues. They can be then sorted from such sources. However, their goal in
Regenerative Medicine is the process of engineering living, functional cell and tissue-based therapies, and
administering them to patients to repair or replace tissue or organ functions lost due to age, disease, damage, or
congenital defects. At the currently available development stage regenerative medicine products and therapies
utilize bio-preservation processes and products in the acquisition of source material, isolation and manipulation
of specific cells, and storage and shipment of a final product dose. To realize the promise of novel cell-based
therapies for pervasive and debilitating diseases, stem cells must be manipulated to isolate them form tissues
and to further select them from others cells, so that they possess the necessary characteristics for successful
differentiation, transplantation, and engraftment. According to NIH’s position
(http://stemcells.nih.gov/info/basics/basics6.asp), to be useful for transplant purposes, stem cells must be
reproducibly made able to:
• Proliferate extensively and generate sufficient quantities of tissue
• Differentiate into the desired cell type(s)
• Survive in the recipient after transplant.
• Integrate into the surrounding tissue after transplant
• Function appropriately for the duration of the recipient's life
• Avoid harming the recipient in any way
Celector® is our novel technology for cell separation that has the key advantage to sort both cells from rough
tissues rough and ex vivo cultured cells without any sort of manipulation. This allows cells maintaining their
native proprieties, and stem cells their potential. No immun0-tagging is required for cell sorting, and the absence
of any type of cell manipulation allows passing regulatory restrictions. Moreover, our tag-less technology allows
for selection/sorting of those stem cell types for which there are not, as yet, efficient technologies on the
market. In fact, fluorescence/magnetic-activated cell sorting (FACS/MACS) technologies do manipulate cells
using immunomarkers, which otherwise might be not available or be poorly specific to efficiently select highly
potent stem cells such as the mesenchymal stem cells (MSCs). Nevertheless, MSCs are among most-promising
adult stem cells for clinical applications.
Novelty and unique features of Celector® then make it a potential “leader tool” among technologies and devices
for cell therapies.
Our technology
Celector® is based on a proprietary technology that isolates and sorts stem cells without immunotags. The novel
methodology selects cells only due to their physical differences: through Celector® even minimal differences in,
for instance, size, shape, rigidity, or membrane conformation can make cells be isolated and sorted into
subpopulations. This absence of immunotagging is the unique feature of Celector®, and it avoids possible cell
manipulation/alteration. This helps cells keeping their native physiology, and for stem cells their regenerative
power. Cells extracted from tissues are introduced in Celector® to isolate stem cells from all other cells
composing the tissue; and also to sort "most potent" stem cells from an ex vivo cultured population, which
ideally is homogeneous in properties. The sorted cells can be further characterized, expanded, or committed to
a specific differentiation for their future use, for instance, for tissue engineering or direct implantation in patients
to reconstruct damaged tissues or for immunomodulation treatments.
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The principles
Stem cells are distributed in all tissues. They can be then sorted from such sources. However, their localization in
each source tissue is not well defined, and they cannot be identified in a specific district isolated from all different
cells, which are more differentiated and originated from the stem cells. Moreover, the lack of homogeneity in
pluri/multipotent SCs severely hinders a definition and standardization for successful stem cell-based therapies.
Finally, cell-type-specific markers such as cell surface proteins are limited known, and they often recognize
multiple members of a SC lineage. Stem cell recovery and functionality are also affected by immunolabeling.
Methods that are less dependent on the identification of particular markers for SC subpopulations, and which
exploit differences in biophysical cell characteristics, are therefore promising when it comes to identify and sort
homogeneous SC subpopulations MSCs are am0ng most-promising adult stem cells for cell therapy applications.
They are adherent, multipotent SCs that are present in quite a few “discarded” tissues. However, MSCs express a
so-rich and diversified panel of surface antigens that limits the possibility to efficiently “distinguish” MSCs on a
immuno-phenotypical basis. To obtain homogeneous stem cells, particularly MSCs, which are properly
characterized, safely usable, and in sufficient number, proper methods of isolation/enrichment/sorting are then
required.
Celector® does operate sorting with no immuno-tagging. Its proprietary separation process exploits differences
in the intrinsic characteristics of the cells, which include size, density and surface properties. Cell sorting occurs
in a biocompatible fluid (PBS-phosphate buffer saline, physiological solutions, culture media) through the sterile
fluidic device that can be disposed once used. The separation process avoid cell contact, and consequent
adhesion on the separation device, and cell-cell aggregation by using in-flow injection of cells and a proprietary
combination of different flow stream rates able to keep cells away from the channel walls, and be swept down
the separation device at different velocities. This can make different cells be collected at different times in
sundry containers.
The flow rate values typically applied guarantee low shear stress on cells. After fractionation is completed, native
physical features then are fully restored. This allows high cell recovery and full maintainance of cell viability and
differentiation features.
How does Celector® look like and work
The Celector® is made as follows:
A. INJECTION SYSTEM
1. Autosampler
2. Pump (PC controlled) and valves
3. Biocompatible fluids for cells separation
B. FRACTIONATION DEVICE
4. Capillaries for fluidic transport of cells to the
separation device and to the detection system
5. Proprietary cell fractionation device using the
proprietary process for cell separation (single or
multi-channel option)
C. DETECTION SYSTEM
6 Optical detection (PC controlled): it counts,
record and recognizes all kind of fractionated cells
D. FRACTION COLLECTOR
7. Fractionated cell collector (PC controlled)
How does Celector® work
1. Autosampler: to be fed to the fractionation device, collects a reproducible numbers of cells.
For single-channel fractionation device: 30 µl < Volume INJECTED < 200 µl (200.000/1.200.000 cells)
For multichannel fractionation device: 500 µl < Volume INJECTED < 3000 µl (3.000.000/9.000.000 cells)
2, 4. Pump, valves and capillaries made of biocompatible material for fluidic management.
3. Fluid reservoirs. (a) Biocompatible fluid for the transport of cells to be separated (mobile phase), (b) Cleaning
solution, (c) Coating solution (biochemical coating of channel internal wall).
5. Capillary fractionation device: where cell separation occurs according to the proprietary method based on the
combined action of the inertial flow of mobile phase and the strength of Earth's gravitational field. This
combination distributes cells at different elevations across the cross section of the channel, depending on the
biophysical properties of cells. The single-channel option provides only one narrow channel while the multi-
channel option provides more channels. Both options can be used in a DISPOSABLE way. The set of channels can
be easily replaced by the user.
6. Proprietary optical detection system. It is interfaced with the fraction collector to select the fractions of cells of
interest.
7. Fraction collector. Cells fractions are collected in tubes, flasks or well plates for specifics use. Labeling tubes
containing the cell fractions is an option to trace the samples.
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Photo preview of cells swimming into Celector® Real time cell counting
Performances in the isolation of human stem cells
A. RESULTS
• Processed cells → umbilical cord, dental pulp MSCs, peripheral blood mononuclear cells, platelets,
leukemia lines, raw cellular samples as lipoaspirates, placenta and whole blood.
• Number of sorted cells → from 3x105 to 106
• Fractionation time → from 10 to 30 minutes.
• Cell purity → highest removal degree of cell clusters , cellular debris and impurities from cellular samples.
• Increased differentiation potential of cells → best cell commitment of the fractionated cells (highest ability
of stem cells to differentiate into specialized cells), also in case of stem cells already isolated by flask
culture (which is the standard procedure to isolate pluripotent cells).
B. COMPARISON WITH ANALOG TECHNIQUES
• Exclusive application → unique technology able to select pluripotent stem cells (e.g. MSCs) without using
immunomarkers.
• No cell manipulation → no immunolabeling and no cell contact with the fractionation device.
• Total biocompatibility → Cells be processed in their own environment (e.g. suspended/diluted in
phosphate buffer or physiological solution).
• User-friendly and fast → short fractionation time (< 20 min), short sample preparation time (only one
centrifugation step, no immunotagging time that usually requires at least 2 hours), automatic, PC-driven
injection, counting and collection of the cells.
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On-progress and future developments
Celector® currently is a fully-functional lab-prototype. Stem Sel is now working in the engineering and
production of the first commercial version, which will be ready to the market on March 2015.
Celector®: current stage and future development
“Lab version”- “Medical/clinical version”
Current Celector® version is targeted to R&D labs (“Lab” version). A “Clinical/Medical” version will be further
developed to scale up the cell production or for direct applications to patients.
A. “Lab version”
Target: LABORATORIES FOR CELL QUALITY CONTROL
1. Research laboratories of public and private institutions.
2. QC laboratories of biotech pharmaceutical industries, cell factories, cell banks, clinics.
B. “Medical version”
Target: CELL BANKS AND CELL FACTORIES
1. GMP-compliant, stem cell production.
C. “Clinical version”
Target: CLINICAL TRIALS IN HOSPITALS, AMBULATORIES AND CLINICS
1. Regenerative surgery
2. Esthetic and reconstructive surgery
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Some applications
Human MSCs (hMSCs) isolated from different tissues exhibit differing lineage-commitment yields, and differing
expression levels of pluripotency markers. This very likely is because of the presence of dissimilar progenitor
cells. This makes difficult to apply techniques based on immunotagging for hMSC sorting. Celector® can be used
to separate hMSCs from potential phenotypically-different contaminants when cells are isolated from clinical
specimens. This allows reducing the number of cell culture passages for hMSC selection, distinguishing hMSCs
derived from different sources, and finally sorting stem cells from an hMSC population isolated from a single
source to obtain the highest differentiation yield.
Characterization of stem cells derived from various sources
hMSCs from various sources and amniotic epithelial cells AECs are fractionated using a specifically developed
protocol. Highly-reproducible fractionations is characteristic for each source, and it could be exploited to
characterize different hMSCs. These different profiles can be correlated with the different differentiation
potential of such cells. The morphology of cell populations from different sources and the relevant fractionation
profile are shown below.
Morphology of cell populations and separation profiles of hMSCs from different sources (BM=bone marrow, AT=adipose
tissue, AM=amniotic membrane, FM=fetal membrane, DP=dental pulp, AEC=amniotic epithelial cell).
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Stem cells sorting from rough clinical samples: fat tissue (lipoaspirate)
Adipose tissue consists of adipocytes and stromal vascular fraction (SVF). In particular, the SVF contains various
hematopoietic, vascular cells and MSCs that are usually identified as adipose-derived stromal (or stem) cells
(ASCs). ASCs from cadaveric donors are isolated by enzymatic digestion with collagenase II.
Starting rough fat tissue sample
Using Celector® SVF is separated to enrich the sample in ASCs with a lower percentage of contaminant cells. In
figure below the results of the separation processes for rough SVF population with the selected fraction F and
for isolated and expanded ASC are shown
The selected cells, named F, have been further characterized observing no differences in morphology, viability,
markers expression, proliferation rate.
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The proliferation rate of Control (unfractionated
expanded ASC) and F cell populations is evaluated
with the Cumulative Population Doubling (CPD)
factor. As shown in the figure on the right or number x,
proliferation index is not modified, thus proving
biocompatibility of Celector®.
The cells are also characterized by cytofluorimetry
using sometypical stem cell markers. The population
of sorted cells F appears more similar to the cells
expanded in culture (ASC) than the raw sample
derived from SVF. This demonstrates an enrichment in
MSCs in F from the rough SVF sample by means of
Celector® sorting.
The immunomodulatory capacity of hMSCs, which is decisive in cell therapies, is analyzed by two assays. In the
first assay, it is evaluated the incorporation of BrdU by peripheral blood mononuclear cells (PBMCs), activated
with the phytohaemagglutin (PHA) and placed in contact with the control raw sample, with ASCs, and with the
cell population sorted by Celector®. In the second assay it is evaluated the PBMCs distribution in the phases of
the cell cycle by flow cytometry analysis.
It is observed that ASCs and F cells are able to arrest the PBMCs in phase GO/G1 with consequent decrease of
BrdU incorporation. This result indicates that selected cells F show immunosuppressive capacity typical for MSCs
confirming that the Celector® separation process doesn’t modify this fundamental native property of stem cells.
This confirms the absence of cell manipulation of the Celector® separation process.
Inhibitory effect of ASCs on the proliferation of PHA-stimulated peripheral blood mononuclear cells (PBMCs) (a). Stimulated
PBMCs are blocked in the G0/G1 phase of the cell cycle when co-cultered with ASCs (b)
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Commitment of hMSCs obtained from SVF to bone tissue immediately after Celector® fractionation is shown
in the following pictures (Control=not induced cells; Unfractionated=non fractionated cells from SVF induced to
bone differentiation; Fractionated=selected fraction F from SVF induced to bone differentiation), and
quantification reported in the plot. The statistical difference between the two induced samples is significant,
using the two-way ANOVA test at 99% of confidence (P <0.01) confirming that Celector® is able to give from
rough clinical specimens an enriched stem cell population bypassing the expansion in culture and making the
differentiation process faster.
Commitments of Celector® treated lipidic hMSCs to other tissues are also demonstrated. In this case different
fractions were collected during the Celector® fractionation (F1, F2, F3), further expanded in culture and induced
to differentiation. Cells from rough tissue ASCs differentiation potential into adipocytes, osteocytes and
chondrocytes is shown for sorted cells and control cells. A representative set of micrographs are reported below.
Adipogenic differentiation: ASCs developed the characteristic lipid vacuoles, stained by Oil Red O incorporation (A).
Osteogenic differentiation: Cells stained with Alizarin Red to detect the newly-synthesized bone matrix, highlighting the
calcium deposits only in differentiated cells (B).
Chondrogenic differentiation: ASCs organized in a microspheroidal (3D pellet cultures), showed the highest content of
total GAGs in differentiated sample, by Blyscan kit staining (C).
In all cases one of the fractions obtained by Celector® treatment shows an higher commitment respect to
unfractionated ASCs, confirming that Celector® is able to sort the most powerful population within an
heterogeneous ones.
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Ex-vivo cultured samples: umbilical cord (Wharton’s Jelly)
Celector® can also isolate cells with different proliferative, differentiative and stemness potential within the
same hMSC population, then sorting different subpopulations. hMSCs derived from umbilical cord connective
tissue (Wharton’s Jelly) (WJ-MSCs) are expanded in culture and then fractionated by Celector®.
The proliferative capacity of the sorted cells (F1-F4) is analyzed by Alamar Blue assay. Looking at the
proliferation plot we can see that a subpopulation, the third fraction (F3) sorted by Celector®, grows faster than
others. This means that Celector® can select within a hMSC population, the subpopulation that owns the best
proliferative potential.
Cells are induced in osteogenic differentiation, and
the result is determinated with Alizarin Red stain.
The Figure shows the different potential of
differentiation along the collected fractions.
Celector® is able to select cells that own the best
stemness properties, in this case the differentiation
potential, or committment.
WJ-MSCs are analysed by flow cytometry for the
expression of the stem cell marker OCT-4 . In the
Figure it can be seen that expression increases with
respect to non fractionated WJ-MSC (NF) cells and
along the different fractions; indicating a stem cell
purification in selected fractions.
“….Talks about Celector®” at the Rizzoli Hospital, Bologna, Italy
Laboratory of Biomechanics & Technological Innovation says: “…..no one actually knows exactly what and how
much you inject in cell treatments, while the quantity and quality of MSCs is a fundamental condition. It’s essential
that cells are not marked. Celector® would have a decisive advantage compared to competitors….”.
Prometeo Laboratory says: “…..using Celector® the quality of cells results better than the ones obtained by
reference techniques using tag or enzymes. When Celector® will be available it will be very interesting tool for tissue
banks…..”.
Ramses Laboratory says: “……whoever does experiments in which cellular response is a very important element
knows that it is essential to maintain cells intact. This is one of the main reasons for the interest in technologies for
tag-less cell enrichment. Application of Celector® to stem cells is significant because all others alternatives are
based on immunomarking. Costs should be an additional advantage of using Celector®…...”
About the Stem Sel Company
Stem Sel is a spinoff of the University of Bologna founded in November 2013 for the commercial exploitation of a
cell selection technology developed by the company founders at the University . The core business activity relies
on the development and commercialization of novel proprietary technologies for highly selective cell selection
and isolation to serve the medical community.
Stem Sel is cooperating with Italian and European
opinion leaders in cell preservation and manipulation
such as cell factories, including a worldwide excellence
centre, the Rizzoli Orthopedic Institute (IOR), which
works on applications for regenerative medicine in
musculoskeletal system. The opinion leaders
collaborating with Stem Sel contribute in developing
and positioning Celector® towards the most mature
applications capable of sustaining the most pressing
needs in the stem cell selection devices sector.
Furthermore they have expressed really positive
opinions and a strong interest in Celector® technology
to the extent that they consider our technology to be
revolutionary compared to present competitors.
Company awards: Stem Sel was initially supported with benefits from (1) Spinner Program of the Region Emilia-
Romagna (2) StartCup, business plan competition won by Stem Sel between 52 projects, selected by investors
and a committee of experts in high technology content companies (3) Intel Business Challenge's European phase
(4) UK Trade Investment's UKIT Entrepreneur Award for business internationalization.
Patents & publications track-record
Patents
The Celector® technology was born from a previous method patented in Italy (IT1371772) and USA
(US12/299350) and with a patent request in progress in Canada (CA2649234). The patents are owned by
University of Bologna, with which Stem Sel has recently come up with an exclusive licence-out agreement.
Patent “Method and device to fractionate stem cells”
Priority date 4th May 2006
Abstract A method and the relevant instrumentation to fractionate living, adherent stem cells,
particularly of human origin, from different sources is disclosed, said method is based on the
non-equilibrium, dynamic fractionation of cells assisted by the Earth gravity field.
Inventors Pierluigi Reschiglian, Barbara Roda, Gian Paolo Bagnara, Andrea Zattoni
Some "celected" publications
“A Tag-Less Method of Sorting Stem Cells from Clinical Specimens and Separating Mesenchymal from
Epithelial Progenitor Cells”. Cytometry Part B (Clinical Cytometry) 76B:285–290 (2009)
Authors:
Barbara Roda, Pierluigi Reschiglian, Andrea Zattoni, Francesco Alviano, Giacomo Lanzoni, Roberta
Costa, Arianna Di Carlo, Cosetta Marchionni, Michele Franchina, Laura Bonsi, and Gian Paolo
Bagnara.
Human lymphocyte sorting by gravitational field-flow fractionation. Anal Bioanal Chem (2008) 392:137–145
Authors: Barbara Roda, Pierluigi Reschiglian, Andrea Zattoni, PierLuigi Tazzari, Marina Buzzi, Francesca
Ricci, Andrea Bontadini.
Gravitational field-flow fractionation of human hemopoietic stem cells. Journal of Chromatography A,
1216 (2009) 9081–9087
Authors: Barbara Roda, Pierluigi Reschiglian, Francesco Alviano, Giacomo Lanzoni, Gian Paolo Bagnara,
Francesca Ricci, Marina Buzzi, PierLuigi Tazzari, Pasqualepaolo Pagliaro, Elisa Michelini, Aldo Roda
A Novel Stem Cell Tag-Less Sorting Method. Stem Cell Rev and Rep (2009) 5:420–427
Authors:
Barbara Roda, Giacomo Lanzoni, Francesco Alviano, Andrea Zattoni, Roberta Costa, Arianna Di
Carlo, Cosetta Marchionni, Michele Franchina, Francesca Ricci, PierLuigi Tazzari, Pasqualepaolo
Pagliaro, Sergio Zaccaria Scalinci, Laura Bonsi, Pierluigi Reschiglian, Gian Paolo Bagnara.
A tag-less method for direct isolation of human umbilical vein endothelial cells by gravitational field-flow
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fractionation. Anal Bioanal Chem (2013) 405:977–984
Authors: Debora Lattuada, Barbara Roda, Chiara Pignatari, Ruben Magni, Federico Colombo, Alessandra
Cattaneo, Andrea Zattoni, Irene Cetin, Pierluigi Reschiglian, Giorgio Bolis.
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Stem Sel s.r.l. - Novel technologies for cell sorting – via Piero Gobetti, 52/3, 40129 Bologna, Italy, / Viale Giuseppe Fanin, 48, 40127 Bologna, Italy VAT code 03331751200 - paid-up capital stock 10.000 €, Business Register: BO-510992 Bank: Cariromagna, IBAN: IT13 B060 1013 2151 0000 0002 560, BIC: IBSPIT2F phone: +39 0514153560/ +39 0516330040 –fax: +39 051 4157907 – pec: [email protected]