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Table 2 Recent studies exploiting feeder-free methods of CRC HAE culture

From: Long-term differentiating primary human airway epithelial cell cultures: how far are we?

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

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