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 esophageal atresia consists in generating tissue constructs for implantation utilizing the patient's own cells. These cells in conjunction with synthetic scaffolding will generate an autologous construct that does not require immune suppression. Some groups have already begun to investigate the use of stem-like cells for esophageal tissue engineering ³ as well as the use of native esophageal epithelial cells to repopulate the mucosa^4-7. Diseases that are present in the esophagus of pediatric patients are often hard to diagnose or study without intervention. Furthermore, utilizing animal models or in vitro immortalized cell line models for pediatric diseases like EoE do not encompass the exact disease pathogenesis or patient specific differences⁸. Therefore, the ability to study a patient's disease process in vitro in order to identify specific disease triggering antigens, evaluate underlying mechanisms and investigate drug treatments would be novel and provide clinicians with information that can aid in patient treatment.

There have been many autologous or patient-specific cell types that have been proposed for use in tissue engineering and studying human disease pathogenesis. However, some of these cell types are limited in their capability to generate enough cells of a specific phenotype to seed a large scaffold or perform high throughput in vitro studies. The use of pluripotent or multipotent stem cells has been the topic of much research discussion, however, limitations and shortcomings for using these cells have been well described⁹. The use of human embryonic stem cells is highly debated and presents many ethical issues. Most importantly, these cells form teratomas, which are similar to a tumor, if they are not differentiated from their pluripotent state prior to delivering them into a living host¹⁰. Furthermore, the use of embryonic stem cells would not be patient-specific and could elicit an allogenic response and the need for immune suppression¹⁰. Induced pluripotent stem cells (iPSCs) are pluripotent cells that can be derived from a patient's own cells. Somatic cells, such as skin cells, can be induced to a pluripotent state using a variety of integrative and non-integrative techniques. These cells then serve as a patient-specific cell sources for tissue engineering or disease investigation. The integration of unwanted genetic material into these cells is a concern many have described and even if sequences are completely removed iPSCs appear to conserve an epigenetic "memory" towards the cell type from which they were derived¹¹. These cells also will form teratomas in vivo if not differentiated prior to transplantation¹¹. Many differentiation protocols have been investigated focusing on epithelial lineages^12,13,14, however, it is very important to note that the cell types resulting at the end of differentiation are not homogenous and only possess a fraction of the cell type of interest. This results in low yield and the need to purify the desired cell type. Although iPSCs are a potential patient-specific cell source, the process to obtain a cell type of interest for either tissue engineering or disease investigation is very inefficient.

Human epithelial cells have been successfully isolated from a variety of both diseased and non-diseased tissues in the human body including: lung¹⁵, breast¹⁶, small intestine¹⁷, colon¹⁸, bladder¹⁹ and esophagus²⁰. It is important to note that human primary cells have a finite number of passages in which the phenotype is maintained^21,22. Unfortunately, this means that the number of cells needed for disease investigation or for seeding an engineered scaffold for implantation may not be achieved. Therefore, new techniques are needed to expand patient cells while still maintaining an epithelial phenotype. Conditional reprogramming of normal and cancerous epithelial cells utilizing feeder cells and ROCK inhibitor was described in 2012 by Liu et al.²³. This technique was utilized to expand cancerous epithelial cells obtained from biopsies of prostate and breast cancer using irradiated feeder cells, ROCK inhibitor and conditional reprogramming medium. The goal was to generate enough cells for in vitro assays such as drug screening. This technique is capable of expanding epithelial cells indefinitely by "reprogramming" these cells to a stem or progenitor-like state, which is highly proliferative. It has been demonstrated that these cells are non-tumorigenic and do not possess the capability to form teratomas^23,24. Furthermore, no chromosomal abnormalities or genetic manipulations were present after passaging these cells in culture using this technique^23,24. Most importantly, these cells are only able to differentiate into the native cell type of interest. Therefore, this technique offers a large reservoir of patient-specific epithelial cells for disease investigation or tissue engineering without the need for immortalization.

Obtaining epithelial tissue from a specific organ in order to study disease processes is often limited and not always possible due to patient risk. For those patients suffering from esophageal disease or defects, endoscopic biopsy retrieval is a minimally invasive approach for obtaining epithelial tissue that can be dissociated and conditionally reprogrammed to provide an indefinite cell source that is specific to the mucosa of that patient's esophagus. This then allows for in vitro studies of the epithelial cells to evaluate disease processes and screen for potential therapeutics. One disease process that could greatly benefit from this approach is Eosinophilic Esophagitis, which has been described as allergic disease of the esophagus⁸. Allergy tests as well as therapeutic approaches could be evaluated in vitro using the patient's own epithelial cells and this data can then be passed onto the treating physician to develop individualized treatment plans. The technique of conditional reprogramming in conjunction with obtaining endoscopic biopsies from pediatric patients offers the ability to expand normal esophageal epithelial cells indefinitely from any patient. This cell source could therefore be teamed together with natural or synthetic scaffolding to provide a patient-specific surgical option for defects, disease or trauma. Having an indefinite cell number would help engineer esophageal constructs that possess a completely reseeded lumen with esophageal epithelial cells in order to help facilitate regeneration of the remaining cell types.

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 mm) and incubate at 37 °C in a cell culture incubator.
   NOTE: These plates can also be made ahead of time and kept in the incubator before use.

3. Making Enzyme Solution for Dissociation

1. Approximately 30 min prior to cell isolation, weigh out 10 units of dispase on a balance based on the units per milligram concentration on the vial. Place the powder in a 15 mL conical tube with 10 mL of Dulbecco's Modified Eagle Medium (DMEM) and place in a 37 °C water bath.

4. Making Cell Culture Medium

1. To make 50 mL of conditional reprogramming medium, mix the following ingredients into a sterile conical tube: 35 mL of F12 (Ham's Medium), 11.5 mL of DMEM, 0.5 mL of L-glutamine, 0.5 mL of 100x penicillin/streptomycin, 2.5 mL of fetal bovine serum (FBS), 250 μg of human insulin, 500 ng of human epidermal growth factor (EGF).
2. To make 500 mL of 3T3 medium, mix 50 mL of FBS, 5 mL of 100x penicillin/streptomycin and 5 mL of L-glutamine with 500 mL of DMEM.
3. To make 500 mL of keratinocyte serum-free medium, thaw EGF and bovine pituitary extract (BPE) supplements (provided with the media). Add the supplements to the 500 mL bottle of base medium and add 5 mL of 100x penicillin/streptomycin

5. Culturing and Irradiating 3T3 Cells as a Feeder Source

1. Prepare 3T3 medium, comprising DMEM, 10% FBS, 1x penicillin/streptomycin and 2 mM L-glutamine.
2. Culture 3T3 cells in tissue-treated T-75 flasks at least 3 days before the arrival of patient samples.
   1. Just prior to the arrival of patient samples, trypsinize 3T3 cells using 5 mL of 0.25% Trypsin-EDTA per flask and incubate at 37 °C for 5 min or until most cells are floating
3. Add 5 mL of 3T3 medium to stop the reaction. Aspirate the cell suspension and transfer to a 15 mL conical tube. Spin for 5 min at 300 x g.
4. Aspirate the medium and resuspend the cells in a defined volume of Trypan blue for cell counting. Combine 10 µL of cells with 10 µL of Trypan blue to exclude dead cells. Load 10 µL of this solution on a hemocytometer for cell counting.
   NOTE: For this protocol, seeding density for irradiated 3T3 cells is 10,900 cells per cm², which is equal to 600,000 cells per 100 mm plate.
5. Aliquot the number of cells necessary to plate a 100 mm plate per patient sample and resuspend in 25 mL of 3T3 medium in a 50 mL conical tube and irradiate with 3000 rads.
6. Following irradiation, spin cells down for 5 min at 300 x g and resuspend in conditional reprogramming medium at 5 mL per 100 mm plate.
7. Set the tube aside until the plating of patient cells takes place later in the protocol (see step 7.6).

6. Obtaining, Preserving and Transporting Pediatric Esophageal Biopsies

1. Add 5 mL of complete keratinocyte serum-free medium with 100 µg/mL  primocin to 15 mL conical tubes and bring it to the medical facility on ice.
   NOTE: These tubes of medium can be stored at 4 °C for up to 2 months.
2. Obtain approximately 8 esophageal biopsies at the time of endoscopy and separate as follows:
   1. Place six biopsies in the 15 mL conical containing complete keratinocyte serum free medium with 100 µg/mL primocin and place on wet ice.
   2. Place one biopsy in 5 mL of buffered formalin and place on wet ice.
   3. Place one biopsy in a small biopsy cryomold with optimal cutting temperature compound.
      1. Immediately drop this mold into a beaker of isopentane placed in liquid nitrogen. Once frozen, place the cryomold in a specimen bag and store on dry ice in a polystyrene foam container.
3. Place the tubes containing biopsies in medium or formalin inside a sealable bag and then into a shipping box containing wet ice. Seal and label the box with a biohazard label and transport it to the laboratory.
   NOTE: The container that contains the dry ice and frozen biopsy is also sealed and labeled with a biohazard label.

7. Processing Patient Tissue for Downstream Culture and Analysis

1. Upon arrival at the laboratory, transfer the frozen biopsy on dry ice to -80 °C freezer for downstream sectioning or RNA extraction. Place the sample in formalin. Store in a 4 °C refrigerator and allow to fix overnight.
2. Spin the remaining biopsies down at 300 x g, and aspirate the medium. Add 10 mL of dispase solution to the conical tube, seal and allow it to incubate at 37 °C in a water bath for 15 min.
3. Following the incubation, spin the biopsies down at 300 x g. Aspirate the medium and resuspend biopsies in 5 mL of 0.05% Trypsin-EDTA.
4. Transfer the 5 mL of trypsin-EDTA containing the biopsies to a 100 mm sterile plate and mince the biopsies using 2 sterilized razor blades as fine as possible.
5. Transfer the 5 mL of trypsin-EDTA containing minced biopsies to a new 15 mL conical. Rinse the plate twice with an additional 5 mL of 0.05% Trypsin-EDTA and add to the conical tube. Incubate the tube for 10 min in a 37 °C water bath.
6. Following the incubation, spin the biopsies down at 300 x g (1.4 RPM) for 5 min and aspirate the medium. Add 5 mL of conditional reprogramming medium to the conical tube as well as the 5 mL of conditional reprogramming medium containing the irradiated feeder cells (see step 5.7).
7. Add ROCK inhibitor (1 μL/mL) to the 10 mL of cell suspension and plate after aspirating the gelatin from the tissue culture dish. Incubate at 37 °C.

8. Culturing and Expanding Cells

1. After approximately 5 days of culture, aspirate the initial medium containing the minced-biopsy pieces and rinse the dish 2-3 times with 5 mL sterile PBS. Add new medium to the plate including ROCK inhibitor at 10 μM.
2. Upon reaching 70% confluency, expand the cells. Add 5 mL of 0.05% Trypsin-EDTA to the plate and incubate in a 37 °C incubator no more than 2.5 min to remove the feeder cells. Following that incubation, aspirate the trypsin-EDTA and rinse the plate with 5 mL of PBS.
3. Aspirate the PBS and add 5 mL of 0.25% Trypsin-EDTA to the plate and incubate for 5-10 min or until the cells are loose from the plate. Add equal amounts of medium to the plate to stop the reaction and transfer all of the fluid to a 50 mL conical tube and spin down at 300 x g for 5 min.
4. Resuspend cells in a defined volume of conditional reprogramming medium and mix 10 μL of the suspension with 10 μL of trypan blue. Count on a hemocytometer to determine total cell number.
5. To expand cells, add 1 million human esophageal cells to a 150 mm plate along with 1.2 million irradiated 3T3 cells in a total volume of 15 mL. Add fresh ROCK inhibitor at 10μM and add the cell suspension to a gelatin-coated 150 mm dish.

9. Freeze and Store Human Esophageal Epithelial Cells

1. To make freezing medium, add 10% DMSO to conditional reprogramming medium. After trypsinizing and counting, aliquot the cells to freeze into a new 50 mL conical tube and spin down at 300 x g for 5 min.
2. Aspirate the medium and add freezing medium at a volume of 2 mL per cryovial. Once the cell suspension is homogenous, aliquot 10⁶ cells in pre-labeled cyrovials. Place in a freezing container at -80 °C for at least 24 h prior to moving the vials to long-term liquid nitrogen storage.

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

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