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Volume 21, No. 6
November, 2014
Cultivated Autologous Limbal Epithelial Cells (CALEC) for the
Treatment of Limbal Stem Cell Deficiency
Milena Di Meo and Myriam Armant
Center for Human Cell Therapy, Boston Children’s Hospital
Boston, MA USA
The limbus, which is anatomically located on the outer edge of the cornea, (Figure 1)
normally contains 5-15% of progenitor cells, called Limbal Stem Cells (LSC) that, under steady
state, contribute to the corneal epithelium homeostasis (Yoon et al., 2014).
Figure 1: The limbus as a source of stem cells involved in corneal homeostasis
Conjunc va
Limbus
Cornea
peripheral
central
Conjunc val epithelial cell
Conjunc val goblet cell
Limbal stem cell (LSC)
Early TAC
Late TAC
Transient
amplifying cell
(TAC)
Post‐mito c cell (PMC)
Terminally differen ated
cell (TDC)
Stra fied epithelial cells
Endothelial cell
See below for
details
Cornea
Anterior chamber
(filled with
aqueous humor)
Lens
Sclera
Vitreous gel
Op c nerve
Epithelial cell
Stromal lamellae
Endothelial cell
Keratocyte
Corneal damages caused by a variety of insults (eye injuries, genetic disorder,
autoimmunity, and inflammation, ocular surface diseases) are often associated with Limbal
Stem Cell Deficiency (LSCD). This condition results in scarring, conjunctivalization and
vascularization of the cornea ultimately leading to progressive vision loss. Corneal
transplantation is not an option in those cases as grafts fail due to lack of host stem and
progenitor cells, which are needed to replenish the surface of the donor graft.
In recent years, cellular therapy such as ex vivo limbal epithelial cell transplantation has
shown promising results as a treatment of unilateral LSCD (Kolli et al., 2010; Marchini et al.,
2012; Nakamura et al., 2004; Rama et al., 2010; Sangwan et al., 2006; Tsai et al., 2000;
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Volume 21, No. 6
November, 2014
Zakaria et al., 2014). However, these clinical trials have not been available in the United States
where LSCD remains an unmet clinical need.
In collaboration with Dr. Ula Jurkanas from the Massachusetts Eye and Ear Infirmary
(MEEI) who will lead the clinical trial, our group at the Center for Human Cell Therapy (CHCT)
developed new methods to yield Cultivated Autologous Limbal Epithelial Cells (CALEC) for the
treatment of unilateral LSCD. The Production Assistance for Cellular Therapies (PACT)
supported this translational project where we developed and validated a 2-stage manufacturing
process (Figure 2). First, enzymatically-isolated limbal epithelial cells are expanded (primary
culture) and then a fraction of the expanded cells (P0) is seeded onto a transplantable substrate
(secondary culture). Among several scaffold options, we opted for AmnioGraft®, which received
tissue designation by the Food and Drug Administration (FDA) and has anti-microbial, antiangiogenic and anti-inflammatory properties.
Figure 2: CALEC process overview
3. Enzyma c diges on
2. Transport to lab
1. Harvest biopsy
4. Pla ng & expansion
End of Primary Culture
5. Harvest & QC
•
•
•
•
Unaffected eye
Counts & viability
Phenotype (iden ty=epithelial)
Colony Forming Efficiency assay
Prolifera ve poten al assay
Start Secondary Culture
CALEC ready for transplant
on the affected eye
6. Seed cells on AmnioGra ®
8. Transport to clinical site
7. Final product QC &
formula on
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November, 2014
Some of the key changes made to the manufacturing included removing the murine
feeder cell line and switching to a serum-free media formulation. In order to mimic as much as
possible the actual clinical trial, we developed an in vitro model of limbal biopsy using cadaveric
corneas. The established manufacturing process is reproducible with a 97.1% success rate
(n=70 products), with an average duration of 18±4 days.
Cellular grafts present unique challenges when it comes to final product testing and
release criteria. Unlike suspension cell-based therapies, samples of the graft cannot be taken
for testing without compromising the integrity of the graft. Our approach was twofold: 1) validate
every steps of the process and 2) develop and validate surrogate assays. We satisfied all
regulatory requirements including defining cell counts and viability. To determine the cell dose
for each graft, we established a method to count cells in situ using a cell-imaging platform. On
the other end, cell viability was determined by validating lactate dehydrogenase (LDH) release
as a surrogate marker. LDH is a cytosolic enzyme that is released in the media when the
plasma membrane is damaged and it is a well-established indicator of cellular toxicity/lysis. We
were able to validate the assay and show a strong correlation (r=0.99) between the amount of
LDH release by cells in the culture supernatant and the percentage of dead cells as determined
by the LIVE/DEAD® assay.
We completed the process and product development phase by working out the postmanufacturing logistics, including final formulation, stability and transport of the cellular graft
back to the clinical site.
The safety and efficacy of CALEC will soon be tested in patients with unilateral LSCD in
a Phase I/II trial as the study (BB-IND-16102) recently cleared the Food and Drug
Administration (FDA).
References
Kolli, S., S. Ahmad, M. Lako, and F. Figueiredo. 2010. Successful clinical
implementation of corneal epithelial stem cell therapy for treatment of unilateral
limbal stem cell deficiency. Stem cells 28:597-610.
Marchini, G., E. Pedrotti, M. Pedrotti, V. Barbaro, E. Di Iorio, S. Ferrari, M. Bertolin, B.
Ferrari, M. Passilongo, A. Fasolo, and D. Ponzin. 2012. Long-term effectiveness
of autologous cultured limbal stem cell grafts in patients with limbal stem cell
deficiency due to chemical burns. Clinical & experimental ophthalmology 40:255267.
Nakamura, T., T. Inatomi, C. Sotozono, N. Koizumi, and S. Kinoshita. 2004. Successful
primary culture and autologous transplantation of corneal limbal epithelial cells
from minimal biopsy for unilateral severe ocular surface disease. Acta
ophthalmologica Scandinavica 82:468-471.
Rama, P., S. Matuska, G. Paganoni, A. Spinelli, M. De Luca, and G. Pellegrini. 2010.
Limbal stem-cell therapy and long-term corneal regeneration. The New England
journal of medicine 363:147-155.
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Sangwan, V.S., H.P. Matalia, G.K. Vemuganti, A. Fatima, G. Ifthekar, S. Singh, R.
Nutheti, and G.N. Rao. 2006. Clinical outcome of autologous cultivated limbal
epithelium transplantation. Indian journal of ophthalmology 54:29-34.
Tsai, R.J., L.M. Li, and J.K. Chen. 2000. Reconstruction of damaged corneas by
transplantation of autologous limbal epithelial cells. The New England journal of
medicine 343:86-93.
Yoon, J.J., S. Ismail, and T. Sherwin. 2014. Limbal stem cells: Central concepts of
corneal epithelial homeostasis. World journal of stem cells 6:391-403.
Zakaria, N., T. Possemiers, S.N. Dhubhghaill, I. Leysen, J. Rozema, C. Koppen, J.P.
Timmermans, Z. Berneman, and M.J. Tassignon. 2014. Results of a phase I/II
clinical trial: standardized, non-xenogenic, cultivated limbal stem cell
transplantation. Journal of translational medicine 12:58.
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