Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Summary

Expansion of human pediatric esophageal epithelial cells utilizing conditional reprogramming provides investigators with a patient-specific population of cells that can be utilized for engineering esophageal constructs for autologous implantation to treat defects or injury and serve as a reservoir for therapeutic screening assays.

Abstract

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

Introduction

Esophageal tissue engineering and eosinophilic esophagitis (EoE) have been the focus of research in many laboratories over the last decade. Congenital defects, such as esophageal atresia, are seen in approximately 1 in 4,000 live births, which results in the incomplete development of the esophagus leading to the inability to eat¹. The incidence and prevalence of EoE have been on the rise ever since the identification of the disease entity in 1993. The incidence of EoE varied from 0.7 to 10/100,000 per person-year and the prevalence ranged from 0.2 to 43/100,000². A new attractive surgical approach to treating long gap es....

Protocol

Esophageal biopsies were obtained after informed consent was obtained from the parents/guardians of the pediatric patients and in accordance with institutional review board (IRB#13-094).

1. Sterilizing Instruments and Gelatin Solution

1. Autoclave forceps, razor blades and scissors prior to handling tissue to prevent contamination.
2. To make 200 mL of 0.1% gelatin solution, combine 200 mL of distilled water with 0.2 g of gelatin. Autoclave and cool prior to use.

2. Coating Tissue Culture Plates

1. Approximately 2 h prior to cell isolation, add enough 0.1 % gelatin to cover a tissue culture dish (100 m....

Results

A summary of the key steps in isolating esophageal epithelial cells from patient biopsies is summarized in Figure 1. Colonies of epithelial cells will form in approximately 4-5 days and will be surrounded by fibroblast feeder cells (Figure 2A). As these colonies expand they will merge with other colonies to form larger colonies (Figure 2B). Once the cultures have become 70% confluent, they need to be expanded (Figure 2C)........

Discussion

The most important steps in order to isolate and expand esophageal epithelial cells from patient biopsies are: 1) adequately dissociating biopsy tissue with minimal cell death; 2) ensure ROCK inhibitor is added to the cell culture medium at every medium change; 3) Do not use more feeder cells than recommended; 4) maintain a clean aseptic culture; and 5) passage cells just prior to reaching confluence.

Due to the patient-related differences in biopsy samples obtained for conditional reprogrammi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Primocin	InVivogen	ant-pm2
Isopentane	Sigma Aldrich	277258-1L
Gelatin From Porcine Skin	Sigma Aldrich	G1890-100G
DMEM	Thermofisher Scientific	11965092
Cryomold	TissueTek	4565
Cryomatrix OCT	Thermofisher Scientific	6769006
15 mL Conical Tubes	Denville Scientific	C1017-p
Complete Keratinocyte Serum Free Medium	Thermofisher Scientific	10724011
Penicillin Streptomycin	Thermofisher Scientific	15140122
Glutamax	Thermofisher Scientific	35050061
Insulin Solution	Sigma Aldrich	I9278-5ml
Human Epidermal Growth Factor (EGF)	Peprotech	AF-100-15
ROCK Inhibitor (Y-27632)	Fisher Scientific	125410
F-12 Medium	Thermofisher Scientific	11765054
Fetal Bovine Serum	Denville Scientific	FB5001
Dispase	Thermofisher Scientific	17105041
0.05% Trypsin-EDTA	Thermofisher Scientific	25300062
0.25% Trypsin-EDTA	Thermofisher Scientific	25200072
100 mm Dishes	Denville Scientific	T1110-20
150 mm Dishes	Denville Scientific	T1115
50 mL Conicals	Denville Scientific	C1062-9
Phosphate Buffered Saline Tablets	Fisher Scientific	BP2944-100
5 mL Pipettes	Fisher Scientific	1367811D
10 mL Pipettes	Fisher Scientific	1367811E
25 mL Pipettes	Fisher Scientific	1367811
9" Pasteur Pipettes	Fisher Scientific	13-678-20D
NIH 3T3 Cells	ATCC	CRL1658