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Intraepidermal Injections of Autologous Epidermal Cell Suspension: A new promising approach to Dermatological Disorders. Preliminary Study

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Intraepidermal Injections of Autologous Epidermal Cell Suspension: A new promising approach to Dermatological Disorders. Preliminary Study

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Regenerative medicine is a modern approach of dermatological treatment, using Epidermal Cells of the interfollicular epidermis (ESCs) for their effect in skin regeneration in chronic ulcers and burns, melanoma, vitiligo, junctional epidermolysis bullosa. Intraepidermal injections of autologous epidermal cell suspension can be a new and very promising treatment for many other cutaneous disorders as non-scarring alopecia (Alopecia Areata, Androgenic Alopecia) or scarring alopecia (Lichern Plano Pilaris alopecia, Discoid Lupus Erithematosus alopecia), anti-aging therapies. The intraepidermal injection of an autologous epidermal cell suspension is a simple, fast and safe surgical procedure: a small, thin portion of the epidermis of the patient undergoes a treatment where a suspension with all the cells collected from the epidermis and cultured for 7 days is injected into the skin. Our preliminary study shows that a suspension contains a signifi cant number of viable cells that survive at day 7 in culture. Our research is ongoing and is focusing on the typing of the different cells in the suspension and evaluation of the presence and the nature of stem cells.
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Journal of Stem Cell Therapy and Transplantation
Open Access
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Research Article
Intraepidermal Injections of Autologous
Epidermal Cell Suspension: A new
promising approach to Dermatological
Disorders. Preliminary Study
Elisa Borsani1,2, Luigi Fabrizio Rodella1,2, Elisabetta Sorbellini3,
Rita Rezzani1,2, Daniela Pinto4, Barbara Marzani4, Giovanna
Tabellini4, Mariangela Rucco3 and Fabio Rinaldi3*
1Department of Clinical and Experimental Sciences, Division of Anatomy and Physiopathology,
University of Brescia, Viale Europa 11, 25123 Brescia, Italy
2Interdipartimental University Center of Research “Adaption and Regeneration of Tissues and
Organs - (ARTO)”, University of Brescia, Viale Europa 11, 25123 Brescia, Italy
3International Hair Research Foundation (IHRF), Milan, Italy
4Department of Molecular and Translational Medicine, Division of Experimental Oncology and
Immunology, University of Brescia, Viale Europa 11, 25123 Brescia, Italy
*Address for Correspondence: Dr. Fabio Rinaldi,
International Hair Research Foundation (IHRF),
Milan, Italy, Tel: 335-1488730; Email:
fabio.rinaldi@studiorinaldi.com
Submitted: 24 December 2017
Approved: 06 December 2017
Published: 07 December 2017
Copyright: 2017 Borsani E, et al. This is
an open access article distributed under the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the
original work is properly cited.
Abbreviations: ESCs: Epidermal Cells of the
Interfollicular Epidermis (ESCs); PRP: Platelet
Rich Plasma (PRP); SCs: Stem Cells
How to cite this article: Borsani E, Rodella LF, Sorbellini E, Rezzani R, Pinto D, et al. Intraepidermal Injections of
Autologous Epidermal Cell Suspension: A new promising approach to Dermatological Disorders. Preliminary Study. J
Stem Cell Ther Transplant. 2017; 1: 066-070.
Introduction
Regenerative medicine is a modern approach of dermatological treatment, using
Epidermal Cells of the interfollicular epidermis (ESCs) to help skin regeneration after
severe damages, such as chronic ulcers and burns [1]. Some clinical trials have already
investigated the use of ESCs to treat other skin diseases such as melanomas [2-5],
Vitiligo [6-10] and Junctional Epidermolysis Bullosa [11].
The intraepidermal injection of an autologous epidermal cell suspension may
be a new and extremely promising treatment for several other skin disorders, such
as non-scarring alopecia (Alopecia Areata, Androgenic Alopecia), scarring alopecia
(Lichen Plano Pilaris Alopecia, Discoid Lupus Erythematosus Alopecia), and anti-aging
therapies. Our experience in autologous preparation like platelet rich plasma (PRP) in
Alopecia Areata [12], a common auto-immune condition which causes hair loss induced
by inlammation and presents limited therapeutic options, has encouraged us in the
Abstract
Regenerative medicine is a modern approach of dermatological treatment, using Epidermal Cells of the
interfollicular epidermis (ESCs) for their effect in skin regeneration in chronic ulcers and burns, melanoma,
vitiligo, junctional epidermolysis bullosa. Intraepidermal injections of autologous epidermal cell suspension can
be a new and very promising treatment for many other cutaneous disorders as non-scarring alopecia (Alopecia
Areata, Androgenic Alopecia) or scarring alopecia (Lichern Plano Pilaris alopecia, Discoid Lupus Erithematosus
alopecia), anti-aging therapies. The intraepidermal injection of an autologous epidermal cell suspension is
a simple, fast and safe surgical procedure: a small, thin portion of the epidermis of the patient undergoes a
treatment where a suspension with all the cells collected from the epidermis and cultured for 7 days is injected
into the skin. Our preliminary study shows that a suspension contains a signi cant number of viable cells that
survive at day 7 in culture.
Our research is ongoing and is focusing on the typing of the different cells in the suspension and evaluation
of the presence and the nature of stem cells.
Intraepidermal Injections of Autologous Epidermal Cell Suspension: A new promising approach to Dermatological Disorders. Preliminary Study
Published: December 07, 2017 67/70
use of autologous derived material for helping skin regeneration and we started to
investigate the effectiveness of epidermal cells against certain speciic skin disorders.
Skin is a complex structure with two different layers, the epidermis and the dermis,
made up of different cell lineages, which ensure the maintenance of the normal skin
homeostasis (tissue repair, barrier function). In the basal layer of the epidermis,
the cell population is heterogeneous as this is made up of several cell types such as
keratinocytes, melanocytes, Langerhans cells, Merkell cells [13]. In the follicular
epidermis, multiple populations of stem cells (SCs) are found in different locations,
with niches of SCs in the basal layer of the epidermis, around the sebaceous gland and
in the bulge niche of hair follicles [14,15].
The intraepidermal injection of an autologous epidermal cell suspension is a
simple, fast and safe surgical procedure: a small, thin portion of the epidermis of the
patient undergoes a treatment where a suspension with all the cells collected from
the epidermis is injected into the skin. The irst step, however, is to select the cells to
be injected. So, the goal of this preliminary study was to investigate the content of an
autologous suspension of epidermal cells and their viability after 7 days of culture and
to ind a simple and fast link between clinical application and in vitro manipulation of
cells. Most interesting, this goal was achieved without using coated lask, as reported
by other authors [16].
Results
Cell morphology
To investigate how cell morphology of epidermal cells in the suspension change
during culturing we observed cell at 0 and 7 day of incubation by means of an inverted
microscope. At day 0, the cells showed a heterogeneous appearance with regard to
shape and size and their nucleus was almost detectable (Figure 1a). At day 7, the cells
maintained a heterogeneous appearance with regard to shape and size, even though
more spheroidal elements were observed. Cells were generally in suspension, only
some microspots of ibroblastoid-shaped cells were well attached to the lask surface
(Figure 1b).
Cytofl uorimetric analysis for cell viability
Cell viability was investigated by means of cytoluorimetric analysis. Representative
dot-plot was reported in igure 2. The number of isolated cells at day 0 is shown in
table 1. At this time point the percentage of viable cells was 59.40% (SD±6.07%),
while the percentage of non-viable cells was 16.48% (SD±5.18%). The remaining cells
formed clusters. At day 7 (data not shown), the percentage of viable cells was 63.55%
(SD±5.41%), while the percentage of non-viable cells was 15.50% (SD±5.68%). The
remaining cells formed clusters. These results show how the absolute percentage of
viable and non-viable cells is maintained after 7 day of culture.
(a) (b)
Figure 1: Epidermal cells at a) day 0 and b) day 7 of culture. White arrows indicate spheroidal cell elements. Scale
Bar=20 um.
Intraepidermal Injections of Autologous Epidermal Cell Suspension: A new promising approach to Dermatological Disorders. Preliminary Study
Published: December 07, 2017 68/70
Discussion
Autologous epidermal cell suspension injected into the skin may be a new approach
to treat several skin disorders. Regenerative medicine could be applied in several
medical ields, and dermatology may use this therapy to successfully treat speciic
disorders. This preliminary study shows that an autologous epidermal cells suspension
contains a signiicant number of viable cells that survive at day 7 in culture.
Our research is currently continuing and it is focusing on the typing of the different
cells in the suspension and evaluating the presence and the nature of stem cells. If
biological studies will also conirm these data, it would be reasonable to think that the
surgical procedure with an autologous epidermal cell suspension may offer several
interesting opportunities for the near future. In summary, this procedure is simple and
fast, lasting 60-90 minutes; there is a very limited risk of side effects (scars in the donor
area, pain in the treated area due to the injections); it is a safe, not presenting risk of
oncogenic disorders and/or immunologic reaction; is not expensive; is an outpatient
treatment. However, some further studies are still needed to validate our indings in
a larger cohort of skin samples and focus on the typing of the cells in the suspension.
For the same reason, the actual effectiveness of this procedure needs to be validated by
some controlled and randomized clinical trials.
Material and Methods
Collection of skin grafts
A graft of skin was collected from 10 healthy volunteers who agreed to undergo the
study. All subjects provided their written informed consent before participating in the
study, and the study was performed according to the Declaration of Helsinki. About
Figure 2: Cyto uorimetric analysis. A representative dot-plot of cell morphology at day 0. Gating cells in P1 are
debris and death cells, gating cells in P2 are viable cells.
Table 1: Number of isolated epidermal cells from 10 donors at day 0.
Subject # Donor surface area (cm2) # epidermal cells (103)
1 5.9 325
2 6.3 475
3 5.7 720
4 6.2 483
5 4.6 700
6 5.3 580
7 6.1 650
8 5.7 734
9 6.2 580
10 4.7 315
average 5.7 556.63
Intraepidermal Injections of Autologous Epidermal Cell Suspension: A new promising approach to Dermatological Disorders. Preliminary Study
Published: December 07, 2017 69/70
six-ten square centimeters of 0.76 mm thick skin (Figure 3) were collected from the
buttock of each subject under local epidermal anesthesia (lidocaine cl 20 mg/ml, 2 ml
per area) through a Zimmer Biomet Electric Dermatome (Zimmer Biomet, Warsaw,
Stati Uniti). The buttock area was chosen to strongly reduce the formation of scars and
cause a milder cosmetic discomfort. Each sample was immediately immersed in a 70%
ethanol solution for 30s to reduce contamination and washed with HBSS twice.
The epidermal grafts were then incubated in a Trypsin solution (Trypsin
0.5g/ EDTA 0.2 solution, Sigma Aldrich, Saint Louis, USA) for 45 minutes at 37°C
(Plasmatherm Barkley, Leopoldshoehe, Germany). After incubation, the Trypsin
solution was discarded and the tissues were washed with HBSS. The epidermis (thin
yellow layer) was treated with a scalpel blade to separate the cells. The supernatant
was suctioned through a sterile syringe and then cultured in Dulbecco′s modiied
Eagle′s medium (DMEM; Sigma Aldrich, Saint Louis, USA) supplemented with 10%
(v/v) heat-inactivated foetal bovine serum (EuroClone, Devon, UK) and 1% penicillin/
streptomycin solution (Sigma Aldrich, Saint Louis, USA) at 37°C in a humidiied
atmosphere of 95% air and 5% CO2. The cells were seeding in a 25 cm2 cell culture
lask at a density of about 20000 cells/cm2. Cell morphology was evaluated before the
seeding and after a 7 day culture by using an inverted Olympus microscope (Olympus
Italia S.R.L., Milan, Italy). The protocols considered previously reported works [16-20].
Cytofl uorimetric analysis for cell viability
Cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was then
removed. The pellet was re-suspended in 3 ml 4% paraformaldehyde in phosphate
buffer 0.1 M pH, 7.4 for ixation. After 20 minutes at +4°C, the cells were centrifuged at
1000 rpm for 5 minutes and the pellet was re-suspended in 500 μl PBS. Finally, the cell
samples were analysed with FACS (BD FACSCanto™ BD Bioscience, San Jose, CA) and
the data were analysed by the BD FACSDiva™ software version 8.8.7 (BD Bioscience,
San Jose, CA). An analysis was performed at day 0 and day 7 after the culture. The
viable cells were identiied using morphological parameters by choosing a range of cell
size combined with morphological cell complexity.
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