We would like to acknowledge Connecticut Children's Medical Center Strategic Research Funding for supporting this work.

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
<ol>
	<li>Autoclave forceps, razor blades and scissors prior to handling tissue to prevent contamination.</li>
	<li>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.</li>
</ol>
2....

<ol>
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The authors declare they have no competing financial interests.

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...

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&#39;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 mucosa4-7. 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&#39;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&#39;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 &#34;memory&#34; 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 lineages12,13,14, 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 maintained21,22. 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 &#34;reprogramming&#34; 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 teratomas23,24. Furthermore, no chromosomal abnormalities or genetic manipulations were present after passaging these cells in culture using this technique23,24. 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&#39;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&#39;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.

<table border="0" cellpadding="0" cellspacing="0" width="843">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Primocin</td>
			<td style="width:172px;">InVivogen</td>
			<td style="width:119px;">ant-pm2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Isopentane</td>
			<td style="width:172px;">Sigma Aldrich</td>
			<td style="width:119px;">277258-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Gelatin From Porcine Skin</td>
			<td style="width:172px;">Sigma Aldrich</td>
			<td style="width:119px;">G1890-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">DMEM</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">11965092</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Cryomold</td>
			<td style="width:172px;">TissueTek</td>
			<td style="width:119px;">4565</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Cryomatrix OCT</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">6769006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">15ml Conical Tubes</td>
			<td style="width:172px;">Denville Scientific</td>
			<td style="width:119px;">C1017-p</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Complete Keratinocyte Serum Free Medium</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">10724011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Penicillin Streptomycin</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Glutamax</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Insulin Solution</td>
			<td style="width:172px;">Sigma Aldrich</td>
			<td style="width:119px;">I9278-5ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Human Epidermal Growth Factor (EGF)</td>
			<td style="width:172px;">Peprotech</td>
			<td style="width:119px;">AF-100-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">ROCK Inhibitor (Y-27632)</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td style="width:119px;">125410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">F-12 Medium&#160;</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">11765054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Fetal Bovine Serum</td>
			<td style="width:172px;">Denville Scientific</td>
			<td style="width:119px;">FB5001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Dispase</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">17105041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">0.05% Trypsin-EDTA</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">25300062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">0.25% Trypsin-EDTA</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">25200072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">100mm Dishes</td>
			<td style="width:172px;">Denville Scientific</td>
			<td style="width:119px;">T1110-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">150mm Dishes</td>
			<td style="width:172px;">Denville Scientific</td>
			<td>T1115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">50ml Conicals</td>
			<td style="width:172px;">Denville Scientific</td>
			<td style="width:119px;">&#160; C1062-9&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Phosphate Buffered Saline Tablets</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td style="width:119px;">BP2944-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">5ml Pipettes</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td>1367811D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">10ml Pipettes</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td>1367811E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">25ml Pipettes</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td>1367811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">9&#34; Pasteur Pipettes</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td style="width:119px;">13-678-20D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">NIH 3T3 Cells</td>
			<td style="width:172px;">ATCC</td>
			<td style="width:119px;">CRL1658</td>
			<td></td>
		</tr>
	</tbody>
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conditional reprogramming of pediatric human esophageal epithelial cells for use in tissue engineering and disease investigation

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.

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)....

Watch this Scientific Journal Video about Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation at JoVE.com

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

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.

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

The overall goal of this procedure is to expand patient-derived esophageal epithelial cells from endoscopic biopsies to study esophageal diseases, or to create a 3D esophageal scaffold for downstream modeling or esophageal tissue replacement. This method can be used to better understand the role of esophageal epithelial cells in esophageal disease. They can also be used to see 3D scaffolds for modeling or implantation.The advantage to this technique is it allows us to generate patient specific epithelial cells from the very small piece of tissue without the need for any genetic or permanent modifications. The implications of this technique extend toward understanding the pathogenesis of eosinophilic esophagitis, as well as developing treatments for esophageal atresia and eosinophilic esophagitis. Generally, individuals new to this technique will struggle with the ability to completely remove the feeder cells prior to harvesting human esophageal epithelial cells in order to maintain purity.Demonstrating this technique will be Christopher Foster, a technician from our laboratory. At least three days before the patient samples are obtained, plate the 3T3 cells in 3T3 culture medium in tissue-treated T75 flasks. On the day of the experiment, just prior to obtaining the samples, detach the 3T3 cells with 5 milliliters of 0.25%Trypsin-EDTA per flask for about five minutes at 37 degrees Celsius.When most of the cells are floating, add 5 milliliters of 3T3 medium to stop the reaction, and transfer the cell suspension into a 15 milliliter conical tube for centrifugation. Re-suspend the pellet in medium for counting. Then, transfer the appropriate number of cells for seeding into a 15 milliliter conical tube.Next, add 25 milliliters of fresh 3T3 medium to the cells for irradiation at 3000 rads. Then, collect the cells by centrifugation, and re-suspend the pellet in conditional programming medium at five milliliters per 100 millimeter plate. After obtaining approximately six esophageal biopsies at the time of endoscopy, place six biopsies in a 15 milliliter conical tube containing complete serum-free keratinocyte medium, supplemented with Primocin on wet ice.Place one biopsy in five milliliters of buffered Formalin on wet ice, and place one biopsy in a small biopsy cryomold containing embedding medium. Immediately immerse the mold in a beaker of ice or pentane inside a container of liquid nitrogen. Once the tissue is frozen, place the cryomold in a specimen bag on dry ice, in a polystyrene foam container.Then, place the tubes of biopsies inside a sealable bag inside a shipping box with the biohazard label containing wet ice, and seal and label both containers with a biohazard sticker for transport to the laboratory. Upon entering the lab, immediately transfer the frozen biopsy to a minus 80 degree Celsius storage for downstream sectioning or RNA extraction. Place the sample in Formalin in a four degree Celsius refrigerator for overnight fixation and place the remaining biopsies in the centrifuge.Replace the medium in each tube with 10 milliliters of dispase solution, and seal the tubes for a 15 minute incubation at 37 degree Celsius in a water bath. At the end of the incubation, spin down the biopsies again, and re-suspend the samples in five milliliters of 0.25%Trypsin-EDTA. Transfer the biopsies to a single 100 milliliter sterile plate, and use two sterilized razors to mince the tissues as finely as possible.When all of the tissue has been cut, transfer the tissue fragments to a new 15 milliliter tube, and rinse the plate two times with five milliliters of 0.05%Trypsin-EDTA. Pool the washes in the tube, and return the samples to a 37 degree Celsius water bath. After 10 minutes, centrifuge the digested sample pieces and the feeder cells, and re-suspend the pellets in five milliliters of conditional reprogramming medium, each.Then, combine the feeder and esophageal biopsy samples into one tube, and add rho kinase, or ROCK inhibitor, to the cells for their co-culture on a gelatin-coated tissue culture dish. After approximately five days, remove the medium and minced biopsy pieces by aspiration, and rinse the dish two to three times with five milliliters of sterile PBS. After the last wash, add 10 milliliters of fresh medium supplemented with ROCK inhibitor, to the plate, and return the co-culture to the incubator until the cells reach 70%confluency.To expand the cells, remove the feeders with five milliliters of 0.05%Trypsin-EDTA at 37 degree Celsius for no more than two and a half minutes. Rinse the plate with five milliliters of fresh PBS to remove the floating feeder cells, and add five milliliters of 0.25%Trypsin-EDTA for a five to 10 minute incubation until the biopsy sample cells are loosened from the bottom of the plate. Then, stop the reaction with 10 milliliters of fresh medium, and transfer the cells into a 50 milliliter conical tube for centrifugation.Re-suspend the pellet in fresh conditional reprogramming medium for counting, and, now, account one times 10 to the sixth human esophageal cells into a new conical tube. Add 1.2 times 10 to the sixth irradiated 3T3 cells to the esophageal cells, and bring the total volume in the tube to 15 milliliters with fresh conditional reprogramming medium. Then, add fresh ROCK inhibitor to the cells, and plate the co-culture onto a gelatin-coated 150 millimeter dish.To freeze the esophageal cells for minus 80 degree Celsius storage, remove the feeders and detach the esophageal cells as just demonstrated. Next, transfer the cells into a new 50 milliliter conical tube for centrifugation, and re-suspend the pellet at a one times 10 to the sixth cells per two milliliter of freezing medium concentration. Then, transfer two milliliters of cells into each pre-labeled cryovial, and store the samples at minus 80 degree Celsius for at least 24 hours before moving them to long-term liquid nitrogen storage.Epithelial cell colonies will form in approximately four to five days, and will be surrounded by fibroblast feeder cells. As these colonies expand, they will merge with other colonies to form larger colonies. Once the culture has become 70%confluent, they need to be expanded.After the feeder cells have been removed, the adherent epithelial cells can be trypsonized and re-plated onto new irradiated feeder cells. Phenotypic characterizati6on of these cells via qRT-PCR reveals an up-regulation of P63, a progenitor cell marker at the end of passage one, compared to the initial biospy sample, indicating that cells have been reprogrammed to a more progenitor stem-like state. When these cells are transferred to normal tissue culture plates, the P63 expression decreases and tight junction protein one, or TJP1, and E-cadherin expressions increase, demonstrating a phenotype shift from progenitor cell to a more differentiated epithelial cell.Flow cytometric analysis of the cells after passage one indicates that greater than 90%of the cells are also positive for EpCAM. Immunofluorescence staining demonstrates that the cells retain their epithelial marker, progenitor marker, proliferation marker, and epithelial tight junction protein expression. The doubling time of esophageal cells from a non-diseased patient has been calculated to be approximately 36 to 40 hours, although it is likely that the doubling time of diseased cells will be slower.Once mastered, this technique should be completed in approximately one hour. While attempting this procedure, be sure to use sterile technique to prevent contamination, and be sure to work expeditiously to maintain cell viability. Following this procedure, other assays, such as transwell culture, can be performed in order to evaluate which antigens are causing esophageal inflammation, or pathogenesis.After development, this assay will pave the way for researchers to identify triggering antigens causing eosinophilic esophagitis, as well as new treatment options for treating these patients. After watching this video, you should have a good understanding of how to isolate and expand esophageal epithelial cells from endoscopic biopsies for use in studying esophageal diseases, or generating 3D models. Don't forget that working with sharp instruments and biological tissues can be hazardous.Be sure to dispose of sharp instruments properly, and use good biosafety practices.

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6.9K Görüntüleme.  UConn Health. The overall goal of this procedure is to expand patient-derived esophageal epithelial cells from endoscopic biopsies to study esophageal diseases, or to create a 3D esophageal scaffold for downstream modeling or esophageal tissue replacement. This method can be used to better understand the role of esophageal epithelial cells in esophageal disease. They can also be used to see 3D scaffolds for modeling or implantation.The advantage to this technique is it allows us to generate patient ...