title: Successful Consecutive Expansion of Limbal Explants Using a Biosafe Culture Medium under Feeder Layer-Free Conditions.
abstract: Transplantation of in vitro cultured limbal epithelial stem cells (LESCs) is a treatment widely used for LESC deficiency. However, the number of limbal tissue donors is limited, and protocols for LESC cultivation often include compounds and/or feeder layers that can induce side effects and/or increase the cost of the culture procedure. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same biopsy using a culture medium in which several potentially harmful compounds were replaced at the same time by biosafe supplements, allowing the LESC cultivation without feeder layers.
  
  
  
  We established feeder layer-free LPCs with three culture media: (1) a modified supplemental hormonal epithelial medium, containing potential harmful components (cholera toxin, dimethylsulfoxide, and fetal bovine serum [FBS]), (2) IOBA-FBS, a medium with FBS but with no other harmful supplements, and (3) IOBA-HS, similar to IOBA-FBS but with human serum instead of FBS. Additionally, the same limbal explant was consecutively cultured with IOBA-HS producing three cultures. LPCs were characterized by real-time reverse transcription polymerase chain reaction and/or immunofluorescence.
  
  
  
  LPCs cultured with the three media under feeder layer-free conditions showed cuboidal cells and no significant differences in the percentage of positive cells for limbal (ABCG2, p63, and K14) and corneal (K3, K12) proteins. Except for ABCG2, the relative mRNA expression of the LESC markers was significantly higher when IOBA-FBS or IOBA-HS was used. LPC1 showed characteristics similar to LPC0, while LPC2 cell morphology became elongated and the expression of some LESC markers was diminished.
  
  
  
  IOBA-HS enables the culturing of up to two biosafe homologous LPCs from one limbal tissue under feeder layer-free conditions. The routine use of this culture medium could improve both the biosafety and the number of available LPCs for potential clinical transplantation, as well as decrease the expense of the culture procedure.
authors.affiliation: a IOBA (Institute of Applied Ophthalmobiology) , University of Valladolid , Valladolid , Spain.|b CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine) , Valladolid , Spain.
authors.forename: Marina
authors.initials: M
authors.lastname: López-Paniagua
authors.affiliation: a IOBA (Institute of Applied Ophthalmobiology) , University of Valladolid , Valladolid , Spain.|b CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine) , Valladolid , Spain.
authors.forename: Teresa
authors.initials: T
authors.lastname: Nieto-Miguel
authors.affiliation: a IOBA (Institute of Applied Ophthalmobiology) , University of Valladolid , Valladolid , Spain.|b CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine) , Valladolid , Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: de la Mata
authors.affiliation: a IOBA (Institute of Applied Ophthalmobiology) , University of Valladolid , Valladolid , Spain.|b CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine) , Valladolid , Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Galindo
authors.affiliation: a IOBA (Institute of Applied Ophthalmobiology) , University of Valladolid , Valladolid , Spain.|b CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine) , Valladolid , Spain.
authors.forename: José M
authors.initials: JM
authors.lastname: Herreras
authors.affiliation: a IOBA (Institute of Applied Ophthalmobiology) , University of Valladolid , Valladolid , Spain.|b CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine) , Valladolid , Spain.
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Corrales
authors.affiliation: a IOBA (Institute of Applied Ophthalmobiology) , University of Valladolid , Valladolid , Spain.|b CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine) , Valladolid , Spain.
authors.forename: Margarita
authors.initials: M
authors.lastname: Calonge
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2 / Q000096:biosynthesis / Q000235:genetics*; D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000378:metabolism; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D003315:Cornea / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D055954:Corneal Endothelial Cell Loss / Q000235:genetics / Q000473:pathology / Q000628:therapy; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D005240:Feasibility Studies; D061252:Feeder Cells; D005786:Gene Expression Regulation; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D008856:Microscopy, Fluorescence; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D012313:RNA / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071999:S100 Calcium-Binding Protein A4 / Q000096:biosynthesis / Q000235:genetics*; D033581:Stem Cell Transplantation / Q000379:methods*; D014019:Tissue Donors
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D015415:Biomarkers; D017077:Culture Media, Conditioned; D009363:Neoplasm Proteins; D000071999:S100 Calcium-Binding Protein A4; C077591:S100A4 protein, human; D012313:RNA
keywords: cell culture; culture media; limbal cells; ocular surface; stem cells