Table 2.
Recent studies exploiting feeder-free methods of CRC HAE culture
| Feeder-free CRC HAE culture | ||||||||
|---|---|---|---|---|---|---|---|---|
| Publication: | Cell type, source, age | Feeder cells? | Expansion: | Differentiation: | Culture length: | Major findings: | Limitations | |
| ONLY ROCKi | Horani [61] | HTE and proximal HBE cells from surgical specimens, expanded and cryopreserved at P1 | NO |
Submerged culture: DMEM/F-12 with 15 mM HEPES, 4 mM L-Glutamine, 3.6 mM NaHCO3, 100 U/mL penicillin, and 100 mg/mL streptomycin with supplements (10 mg/mL insulin, 5 mg/mL transferrin, 0.1 mg/mL cholera toxin, 25 ng/mL EGF, 30 mg/mL BPE, 5% FBS (v/v)) Vessel coating: rat tail collagen type I (50 mg/ml) |
In ALI, mTEC/Basic Medium with 2% NuSerum and retinoic acid [54] |
Highest proliferation at 5 µM Y27632. ~ 25% cilia in hTEC | 1. Y27632 increased cell proliferation, efficiency of lentiviral transduction (up to 80%), and facilitated antibiotic selection of transduced cells | Decreased number of goblet cells during ALI (0–19 Muc5AC + cells/ 100-power visual field) |
| Jonsdottir [68] | Fresh HBE cells (bronchoscopy or surgical samples), adult patients | NO |
Submerged culture: BEGM medium with 10uM Y-27632 Vessel coating: Collagen Type IV |
ALI, LHC:DMEM medium + supplements [33] Vessel coating: Collagen Type IV |
Cells used until P4 |
1. Lentiviral suspension transduction method established (15 -70% efficiency, depending on the virus titer) 2. Addition of ROCKi slightly decreased this efficiency (5%–10% lower), but allowed selection of cells after transduction |
Not all constructs were equally effective—careful evaluation and testing of the viral constructs is required | |
| OTHER SUPPLEMENTS/ INHIBITORS | Mou et al. [71] | fresh human trachea, bronchi, BAL, or induced sputum | NO |
Submerged culture: Different media (with/without 5–10 mM ROCKi, 0.5–1 mM A-83–01, 0.5–1 mM DMH-1, and 1 mM CHIR99021, separately or in various combinations). Media tested: SAGM (Lonza), HTEC (You and Brody, 2013), BEGM (Fulcher et al. 2005), LHC (Life Technologies), LHC-9 (Invitrogen), and AECBM (from ATCC and PromoCell) Vessel coating: laminin-enriched 804G-conditioned medium |
ALI: Pneumacult ALI medium |
Cell divisions until P25-P30 (PD > 40) Goblet cell differentiation until P25, but efficient ciliation until P10 Physiological CFTR reactions—until P8 TEER – stable |
1. Expansion possible up to 25 passages (PD 40) 2. Expansion is efficient (from 1–20 *103 cells to ~ 1 × 10 15 cells at P10—in 50 days) 3) BAL or induced sputum samples (< 2000 cells) expanded to 109 or 1010 within 1 month |
Resemblance to native epithelium decreases with time (CFTR reactions only until P8, mucociliary differentiation until P10) 3) Culture from induced sputum samples less effective than from BAL |
| OTHER SUPPLEMENTS/ INHIBITORS | Zhang et al. [72] | HBE cells | NO |
Submerged culture: KSFM with 1 µM A83-01, 5 µM Y-27632, (KSFM + A + Y) Vessel coating: collagen I |
ALI, Filter seeding in EpiX + 1.5 mM CaCl2. After confluence, basolaterally Pneumacult-ALI medium |
EpiX medium supports efficient HBECs expansion and differentiation until 45–60 PDs (P12–P16) |
1. Increased cell yield (> 50 mln cells from CF patients in 3–4 weeks; > 1 * 106-fold increase) 2. Retained genome integrity, no tumorigenicity 3. Cells have low stress levels 4. Retained CFTR response > 30 PDs (reduced, but exists); 5. Single cell cloning possible |
Large cells accumulated slowly over passages and eventually the majority of the population appeared senescent or differentiated |
| Lu et al. [74] | tracheal neonatal aspirates (< 100 epithelial cells/ sample) | NO |
Submerged culture: SAEG with A-8301 (1 μM) and Y27632 (5 μM), rapamycin (5 nM) Vessel coating: N/A |
ALI Filter seeding: growth medium, Pneumacult-ALI and DMEM with 4-(2-hydroxyethyl)-1-piperazineethaNesulfonic acid (2:1:1 ratio) 12 h after filter seeding: complete Pneumacult-ALI medium (submerged, ALI from day 1) |
Expansion for at least P15 (40–50 PDs) Cells retain BC markers (NGFR, PDPN) Differentiation until P16 |
1. Efficient generation of cultures from neonatal tracheal aspirates 2. Efficiency of mucociliary differentiation comparable to other methods |
Abrupt halt of proliferation at the end of the culture– probably due to telomere erosion | |
| Koh et al. [67] | HBE cells, from leftover transplant | NO |
Submerged culture: BEGM + 10 uM Y-27632 Vessel coating: human placental collagen |
Medium: LHC:DMEM mixture with supplements [33] | N/A | Double nucleofection allowed up to 100% targeting without antibiotic selection | ||