Optimal isolation and xeno-free culture conditions for limbal stem cell function

Abstract

Purpose: To preserve limbal stem cell (LSC) function in vitro with xenobiotic-free culture conditions.

Methods: Limbal epithelial cells were isolated from 139 donors using 15 variations of three dissociation solutions. All culture conditions were compared to the baseline condition of murine 3T3-J3 feeders with xenobiotic (Xeno) keratinocyte growth medium at 20% O2. Five Xeno and Xeno-free media with increasing concentrations of calcium and epidermal growth factor (EGF) were evaluated at 5%, 14%, and 20% O2. Human MRC-5, dermal (fetal, neonatal, or adult), and limbal stromal fibroblasts were compared. Statistical analysis was performed on the number of maximum serial weekly passages, percentage of aborted colonies, colony-forming efficiency (CFE), p63α(bright) cells, and RT-PCR ratio of p63α/K12. Immunocytochemistry and RT-PCR for p63α, ABCG2, Bmi1, C/EBPδ , K12, and MUC1 were performed to evaluate phenotype.

Results: Dispase/TrypLE was the isolation method that consistently showed the best yield, viability, and CFE. On 3T3-J2 feeders, Xeno-free medium with calcium 0.1 mM and EGF 10 ng/mL at 20% O2 supported more passages with equivalent percentage of aborted colonies, p63α(bright) cells, and p63α/K12 RT-PCR ratio compared to baseline Xeno-media. With this Xeno-free medium, MRC-5 feeders showed the best performance, followed by fetal, neonatal, adult HDF, and limbal fibroblasts. MRC-5 feeders supported serial passages with sustained high expression of progenitor cell markers at levels as robust as the baseline condition without significant difference between 20% and 5% O2.

Conclusions: The LSC function can be maintained in vitro under appropriate Xeno-free conditions.

Keywords: cell culture; limbal; oxygen; stem; xeno-free.

Figure 1

Strategy for selection of optimum isolation method and Xeno-free culture conditions. LECs were isolated and dissociated into a single cell suspension that was seeded on feeder cells and cultured in parallel under different conditions. The colonies formed were stained bright pink with rhodamine for identification and counting. ICC, immunocytochemistry (number of p63α^bright cells); K12, cornea differentiation marker cytokeratin 12.

Figure 2

Selection of isolation method based on yield and CFE. Results of six selected isolation methods (Table 1) of the total of 15 variations are shown, three of them (numbers 1–3) reported previously and three variations of method number 3 (numbers 4–6). They were evaluated for yield as total number of cells isolated from a cadaveric donor corneoscleral rim (A) and for CFE on 3T3-J2 feeders (B). Each bar represents mean and SEM from 6 to 15 different limbal specimens derived from a total of 139 donors. Statistically significant difference of ***P = 0.0001 or ****P = 0.00001 with ANOVA/Bonferroni tests between methods 5, 6, and methods 1 to 3 in (A), and methods 1 to 4 in (B). Method 6 (Dispase 2.4 IU/mL for 2 hours and TLE for 10 minutes) consistently showed the best results.

Figure 3

Evaluation of Xeno-Free media for growth of LECs. X-KGM medium was selected as the baseline medium, included in every experiment, and dissociated LECs (method 6) were seeded at the same density and grown in parallel. XF-Ca0.1 medium supported more weekly passages to senescence than any other medium (A), while it showed low percentage of aborted colonies (B), and not significantly different CFE and HFE (C) compared to the baseline medium. Low calcium (0.1 mM) Xeno-free medium supported more passages than higher calcium media, all with EGF 10 ng/mL (D). The XF-Ca0.1 media supported more passages when supplemented with 10 to 20 ng/mL EGF (E). Immunocytochemistry for p63α^bright cells, quantified and normalized for baseline medium X-KGM, showed significantly higher expression in low calcium Xeno-free medium and lower expression in X-MCBD medium (F). The RT-PCR ratio of p63α/K12 normalized for X-KGM was not significantly different among the different media (G). Each bar represents mean and SEM from 6 to 23 fresh limbal specimens. Differences of ***P = 0.0001, **P = 0.009, and *P = 0.01 or 0.019 with ANOVA between all media variations in all oxygen tensions (20%, 14%, and 5%), but for simplicity results from only 20% O₂ are shown in this figure. Abbreviations of media names as in Table 2. Xeno-free low calcium medium with EGF 10 ng/mL showed the best performance.

Figure 4

Effect of oxygen tension on LEC culture. Xeno-free medium with calcium 0.1 mM and EGF 10 ng/mL supported more weekly passages of LECs to senescence under 20% O₂, while X-KGM performed better under 5% O₂ (A). Immunocytochemistry for p63α^bright quantified and normalized for baseline medium X-KGM at 20% O₂ showed higher expression in the Xeno-free medium under 20% O₂ (B). The RT-PCR ratio of p63α/K12 normalized for X-KGM at 20% O₂ was not significantly different among the different media and O₂ tensions (C). Each bar represents mean and SEM from 6 to 23 fresh limbal specimens. Difference of ***P = 0.0001 and *P = 0.01 with ANOVA, between all media variations in all oxygen tensions (20%, 14%, and 5%), but for simplicity results from these two media are shown in this figure.

Figure 5

Comparison of human feeders with Xeno and Xeno-free media. Human lung fibroblasts MRC-5 and HDF, either F-HDF, N-HDF, or A-HDF feeders, supported LEC cultures with low percentage of aborted colonies (A), high expression of p63α^bright cells by immunocytochemistry (B), and high RT-PCR ratio of normalized p63α/K12 expression (C), with X-KGM and with low calcium (0.1 mM) Xeno-free (XF) medium with EGF 10 ng/mL. Each bar represents mean and SEM from 6 to 14 fresh limbal specimens, normalized to the baseline condition of mouse 3T3-J2 feeders with X-KGM medium. Significant differences of ***P = 0.0001 for aborted colonies, *P = 0.022 for ICC and *P = 0.03 for RT-PCR with ANOVA tests. The MRC-5 and F-HDF human feeders were selected for further evaluation.

Figure 6

Further evaluation of human fibroblast feeders MRC-5 and F-HDF with Xeno-free medium. Human lung fibroblasts MRC-5 and F-HDF feeders supported LEC cultures of different morphology at first passage; colonies looked more round and separated from the 3T3-J2 or MRC-5 feeders with either Xeno or Xeno-free medium, while F-HDF feeders supported colonies that were more difficult to identify among the feeders (×5, tissue culture phase microscope, [A] first column). In higher magnification (×20), phase photographs and immunocytochemistry for progenitor cell marker p63α (red), with nuclear counterstain DAPI (blue) overlay ([A], right column) showed presence of limbal progenitor cells growing in more (3T3-J2 or MRC-5) or less (F-HDF) distinct colonies ([A], right column). The RT-PCR ratio of p63α/K12 showed that MRC-5 feeders with XF medium could support favorable ratio for progenitor cell expansion comparable to 3T3-J2 feeders with X-KGM medium for multiple weekly passages (B). Points represent mean and SEM of four replicates, normalized to isolated limbal epithelial cells before seeding (P0) in parallel in the four different conditions under 20% O₂. Xeno-free (XF) medium had calcium 0.1 mM and EGF 10 ng/mL.

Figure 7

Limbal epithelial cells express appropriate markers of LSCs in multiple passages under Xeno-free culture conditions. Colonies at passage 7 expressed limbal progenitor cell marker p63α or p63 (red, [A–D]) as well as ABCG2 (green, [A]), C/EBPδ (green, [B]), cytokeratin K15 (K15, green, [C]), and cornea differentiation marker cytokeratin 12 (K12, green, [D]), and were counterstained with DAPI (blue, [A–D]) for overlays ([A–D]). The RT-PCR expression (mean and SEM from 4 replicates) of limbal progenitor cell markers p63α, ABCG2, Bmi1, C/EBPδ , and differentiation marker K12, was normalized to isolated limbal epithelial cells before seeding (P0), and was preserved for multiple weekly passages (E).