Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

Summary

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Abstract

The incidence of both esophageal adenocarcinoma and its precursor, Barrett’s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.

A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.

This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett’s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

Introduction

The esophageal mucosa comprises a stratified, squamous epithelium above a layer of connective tissue, the lamina propria, and is one of the first sites to encounter ingested environmental stressors. Exposure to dietary toxins is implicated in the development of esophageal squamous carcinoma, while duodenogastro-esophageal reflux is a critical factor in the pathogenesis of Barrett’s Metaplasia, which is associated with increased risk of progression to esophageal adenocarcinoma. Esophageal carcinomas are the 8^th most common malignant tumor in UK males and esophageal adenocarcinoma is rapidly increasing in the Western world¹. Furthermore, there has been little improvement in disease prognosis, with an overall 5-year survival rate of around 15%. Consequently there is a need for experimental platforms to investigate the impact of exposure to environmental stressors on this esophageal epithelium and their potential involvement in the development of metaplasia or neoplasia.

Although immortalized or tumor cell lines allow researchers to study the response of epithelial cells to these stressors in vitro, they remain proliferative and fail to differentiate into the mature epithelial cells found on the uppermost layers of the esophageal mucosa. Furthermore, cells lines that have already undergone tumorigenesis may provide only limited information regarding the initial responses of normal cells within the epithelium to environmental factors; and this is the stage when the potential for therapeutic intervention may be highest. Finally, conventional cell culture systems fail to capture the potentially important interactions between epithelial and mesenchymal cells and between these cells and the surrounding matrix that occur within tissues in vivo.

Animal models provide a more realistic microenvironment for studying the responses of the esophageal epithelium and can incorporate the artificial induction of gastro-esophageal reflux disease². However it can be more challenging to manipulate the environmental stressors in these models and they may not fully represent the response within the human esophagus.

Other experimental human esophageal models have been developed that utilize primary cells, immortalized cells or tumor cell lines on a collagen, or combined collagen/Matrigel, scaffold containing fibroblasts^3,4. It is less labor intensive to generate these scaffolds than the acellular esophageal scaffold described in this manuscript, and these organotypic models provide a useful tool, particularly in the study of tumor invasion^5,6, where tumor cell infiltration into the collagen gel can be readily observed. However these collagen gels have non-native mechanical properties and lack certain features of the original tissue, including a specific basement membrane and the appropriate surface topography. This can influence the behavior of cells resulting in, for example, poorer adhesion between the epithelium and scaffold when using a collagen gel scaffold⁷. As a consequence the acellular porcine esophageal scaffold was developed, with the advantage of being a more biologically realistic scaffold and thus more appropriate for use as an experimental platform. It has also been shown that it is better to incorporate primary cells into the esophageal constructs than immortalized esophageal epithelial cell lines, such as Het-1A, since these cells form a multi-layered epithelium but fail to stratify or differentiate^4,7,8.

Consequently, this protocol has been adapted from a method already in use in the MacNeil laboratory for making tissue engineered skin and oral mucosa^9,10 and incorporates a de-cellularized porcine esophageal scaffold combined with primary human esophageal epithelial cells and fibroblasts. This protocol produces a mature, stratified epithelium, similar to that of the normal human esophagus as demonstrated by CK4, CK14, Ki67 and involucrin staining. The resulting model provides an experimental platform to study responses to environmental stressors, and has been used effectively to investigate changes in gene expression in the esophageal epithelium in response to refluxate components¹¹.

Protocol

Human esophageal cells are obtained from patients undergoing gastric or esophageal surgery. Informed consent is obtained for the tissue to be used for research purposes, and the tissue used anonymously under the appropriate ethical approvals (SSREC 165/03, Human Research Tissue Bank Licence 12179).

1. Isolation of Human Esophageal Epithelial Cells

1. Working in a laminar flow tissue culture hood and using sterile technique, prepare the epithelial culture medium by mixing Dulbecco’s Modified Eagle’s medium (DMEM) and Ham’s F12 in a 3:1 ratio (v:v). Add 10% (v/v) FCS, 10 ng/ml epidermal growth factor, 0.4 µg/ml hydrocortisone, 1.8 x 10^-4 M adenine, 5 µg/ml insulin, 5 µg/ml transferrin, 2 x 10^-7 M triiodothyronine, 1 x 10^-10 M cholera toxin, 2 x 10^-3 M glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.625 µg/ml amphotericin B.
2. Using standard sterile techniques, put 11 ml of DMEM, 10% FCS, 2 x 10^-3 M L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.625 µg/ml amphotericin B into a T75 tissue culture flask. Add 1 x 10⁶ lethally irradiated mouse 3T3 fibroblasts¹² in 1 ml of the same medium and incubate overnight at 37 °C in a 5% CO₂, humidified atmosphere. This will produce a feeder layer for the subsequent culture of epithelial cells.
3. Using a scalpel and following the recommendations of a histopathologist, dissect an approximately 2 cm² sample of tissue from the disease-free background region of esophageal squamous mucosa obtained from patients undergoing gastric or esophageal surgery. Transport to the laboratory in a 50 ml container of sterile transport medium (PBS 100 IU/ml penicillin, 100 µg/ml streptomycin and 0.625 µg/ml amphotericin B) at room temperature.
4. At this point, store the tissue in the transport medium for up to 12 hr (overnight) at 4 °C, for convenience; however do not use longer storage periods as they result in reduced cell viability.
5. Working in a laminar flow tissue culture hood and using sterile technique, sterilize a scalpel blade by soaking in 70% ethanol for 5 min. Rinse in sterile PBS.
6. Place the tissue in a sterile Petri dish and use the scalpel to cut it into 0.5 cm strips. Add 5 ml of 0.1% (w/v) trypsin in PBS to the Petri dish, place the lid on the dish and incubate for 1 hr at 37 °C.
7. Add 5 ml of FCS to the tissue in the Petri dish to inhibit the trypsin. Holding the strip of tissue with sterile forceps, gently scrape the epithelial surface with the scalpel blade for 1-2 min per strip to remove the epithelial cells into the surrounding medium. Remove the scraped tissue and set aside. Repeat the process for all the tissue strips.
8. Collect the medium containing the detached epithelial cells into a 15 ml centrifuge tube and centrifuge (5 min, 200 x g). Pour off the supernatant and resuspend the pellet in 12 ml of epithelial medium (described in step 1.1).
9. Pour off and discard the medium from the feeder cell layer in the T75 flask prepared in step 1.2. Use a sterile pipette to add the cell suspension containing the freshly isolated epithelial cells to the flask and incubate at 37 °C in a 5% CO₂, humidified atmosphere.
10. After 24 hr pour off and discard the culture medium, which will also contain cell debris, and replace with 12 ml of fresh epithelial medium. Continue incubating at 37 °C in a 5% CO₂, humidified atmosphere. Colonies will start to appear over the next 24 to 48 hr and can be viewed by placing the culture flask under a standard light microscope. Pour off the spent culture medium and replace with an equal volume of fresh medium every 2 to 3 days.
11. Once the flask has reached 80% confluency, passage the cells¹³. Split the cells in a ratio of 1:5 to increase cell numbers for use in the model. Follow step 1.2 to establish feeder layers of irradiated 3T3 cells in each flask that will be receiving epithelial cells 24 hr prior to passaging the cells.
    Note: Use the epithelial cells in the esophageal model described below between passages 1 and 4 only.

2. Isolation of Human Esophageal Fibroblasts

1. Working in a laminar flow cabinet and using sterile technique, take the tissue set aside in step 1.7. Place in a sterile Petri dish and mince finely by chopping with a sterile scalpel blade. Add 10 ml of 0.5% (w/v) collagenase A solution and place the lid on the Petri dish and incubate at 37 °C overnight in a 5% CO₂, humidified atmosphere.
2. Transfer the digest to a 15 ml centrifuge tube and centrifuge (10 min, 200 x g), carefully pour off and discard the supernatant and resuspend the pellet in 10 ml of fibroblast culture medium (DMEM 10% FCS, 2 x 10^-3 M L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.625 µg/ml amphotericin B).
3. Place the suspension into a T75 flask and incubate at 37 °C in a 5% CO₂, humidified atmosphere. After 24 hr pour off the medium, which will contain debris and replace with 12 ml of fresh fibroblast medium. Replace the culture medium every 2 to 3 days and passage cells once they have reached 80% confluency¹³, splitting the cells in a 1:5 ratio to increase cell numbers.
   Note: Use the fibroblast cells in the model described below between passages 4 and 10 only.

3. Preparation of the De-cellularized Esophageal Scaffold

1. Obtain intact porcine esophagi at an abattoir from freshly slaughtered Landrace pigs that are used for food production. One single esophagus will provide sufficient scaffolds for approximately 30 separate constructs.
   Note: Due to the extensive and time consuming sterilization protocol required, it is normally worth obtaining and processing a minimum of 10 esophagi at one time.
2. Immediately place the freshly excised esophagus into a 180 ml sterile pot containing approximately 150 ml of 10% povidone-iodine solution for 5 min to reduce any contaminating microbial load. Transfer the esophagus to a second pot containing approximately 100 ml of sterile PBS containing 200 IU/ml penicillin, 200 µg/ml streptomycin, and 1.25 µg/ml amphotericin B.
   1. Replace the lid on the container and transport the esophagi back to the laboratory at room temperature. Two or three esophagi can be transported in each container, depending upon their size.
3. Once at the laboratory, handle the tissue in a laminar flow hood using aseptic technique to minimize microbial contamination. Sterilize tweezers, scissors and scalpel blades by soaking for 5 min in 70% ethanol, and rinsing in sterile PBS.
4. Handle the esophagus using sterile tweezers. Using the scissors, cut open the esophagus longitudinally. Rinse the tissue by placing the esophagus into a 180 ml sterile pot containing approximately 100 ml of sterile PBS and shaking gently to remove any debris.
5. Clean a cork dissection board by spraying liberally with 70% ethanol and allow to dry.
6. Remove the esophagus from the PBS, and using sterile needles, pin out the esophagus onto the board, mucosal surface uppermost. Grasp the mucosal surface at one end of the esophagus with sterile tweezers and pull away from the board. This will separate the mucosa from the underlying submucosa.
   1. Then, use a sterile scalpel blade to dissect the esophagus longitudinally along the lamina propria, removing pins as the dissection progresses to allow the mucosa to be lifted away.
7. Retain the dissected mucosa and discard the underlying submucosa.
8. Cut off and discard the most proximal and distal 2 cm of the mucosa as there is a greater number of submucosal glands and a thicker lamina propria in those areas, respectively⁷.
9. Use a scalpel to cut the remaining tissue into 5 cm². Place all the cut tissue into a 180 ml sterile pot containing approximately 150 ml of sterile PBS and agitate gently to rinse the tissue. If processing more than five esophagi at one time it may be necessary to use multiple pots for this and the three subsequent steps.
10. Using sterile tweezers, transfer the cut tissue into a 180 ml pot containing 150 ml of sterile 1 M NaCl, 200 IU/ml penicillin, 200 µg/ml streptomycin and 1.25 µg/ml amphotericin B. Incubate for 72 hr at 37 °C.
11. Place the tissue into a fresh 180 ml pot containing 150 ml sterile PBS. The epithelium will have begun to visibly separate from the underlying tissue. Gently peel the detaching epithelium from the pig esophagus using sterile forceps and discard.
    1. Place the remaining tissue into a fresh 180 ml pot and add 150 ml of sterile PBS. Agitate gently for 5 min to wash. Pour off the PBS and repeat a further two times with fresh PBS.
12. Place all the tissue into a 500 ml glass bottle containing 400 ml of sterile 80% (v/v) glycerol solution for 24 hr at room temperature. Transfer to 400 ml of sterile 90% (v/v) glycerol solution for a further 24 hr.
13. Store in 400 ml of sterile 100% glycerol solution at room temperature for a minimum of 4 months to dehydrate and sterilize the tissue while preserving the integrity of the basement membrane.

4. Production of the Culture Media

1. Prepare chelated newborn calf serum for use in the culture medium.
   1. Pour 100 g of Chelex 100 into a glass 1 L bottle and add de-ionized water. The exact volume is not critical but sufficient water must be added to cover the Chelex powder. Add a magnetic stirrer and sterilize by autoclaving (121 °C for 15 min).
   2. Allow the bottle to cool and the Chelex to settle. In a laminar flow cabinet pour off the excess water, add 500 ml of newborn calf serum (NCS) and leave to stir overnight at 4 °C.
   3. Stop stirring and allow the Chelex to settle, this can take up to 30 min. In a laminar flow cabinet decant the chelated serum using a sterile pipette, taking care not to disturb the Chelex, and store in 10 ml aliquots at -20 °C.
2. Prepare three different media in a laminar flow cabinet, commencing with pro-proliferative and ending with pro-differentiative formulations.
   1. Prepare Composite Medium I comprised of DMEM and Hams F12 in a 3:1 ratio (v:v), 4 x 10^-3 M L-glutamine, 0.5 µg/ml hydrocortisone, 1 x 10^-4 M O-phosphorylethanolamine, 2 x 10^-12 M triiodothyronine, 1.8 x 10^-4 M adenine, 1.88 x 10^-3 M CaCl₂, 4 x 10^-12 M progesterone, 10 µg/ml insulin, 10 µg/ml transferrin, 10 ng/ml selenium, 1 x 10^-3 M ethanolamine and 0.1% (v/v) chelated NCS.
   2. Prepare Composite Medium II as Composite Medium I except replace chelated serum with 0.1% (v/v) non-chelated NCS.
   3. Prepare Composite Medium III as Composite Medium II except omit progesterone and increase serum to 2% (v/v) non-chelated NCS.

5. Production of the Human Esophageal Mucosa Model

1. Perform all work in a laminar flow hood, using aseptic technique to maintain a sterile environment. Sterilize all tools by soaking in 70% ethanol for 5 min, before rinsing in sterile PBS.
2. The day before it is required, rehydrate the acellular porcine esophageal scaffold. One piece of scaffold is required for each construct to be prepared.
   1. To rehydrate the tissue place sufficient pieces of porcine scaffold into a pot containing 100 ml of sterile PBS, agitate and soak for 10 min. Decant the PBS and replace with fresh PBS. Repeat the washing process five times to ensure removal of all glycerol traces.
3. Test the scaffold sterility by incubating the rehydrated tissue overnight in 100 ml of DMEM at 37 °C. If there is no change to the color or turbidity of the culture medium at the end of the incubation period the scaffold is assumed to be sterile. Once sterility has been confirmed, place the 5 cm² acellular scaffold into a 6 well plate, submucosal side uppermost.
4. Place a sterile medical grade stainless steel ring (internal diameter 10 mm, external diameter 20 mm) onto the center of each scaffold and gently press down using sterile forceps, to ensure an adequate seal with the tissue.
5. Harvest the fibroblasts using Trypsin-EDTA¹³ to produce a cell suspension. Count the cells using a haemocytometer¹⁴. Centrifuge the cell suspension (10 min, 200 x g). Resuspend the cell pellet into an appropriate volume of fibroblast medium to produce a final cell count of 2.5 x 10⁶ cells per ml.
6. Add 0.2 ml of fibroblast medium, containing 5 x 10⁵ human esophageal fibroblasts, within each ring. Flood the area around the outside of the ring with approximately 2 ml of fibroblast medium. Incubate at 37 °C in a 5% CO₂, humidified atmosphere.
7. After 24 hr remove the rings and fibroblast medium and add at least 5 ml of fresh fibroblast medium to each well, ensuring that the scaffold is totally immersed in medium. Culture for 1 week, at 37 °C in a 5% CO₂, humidified atmosphere replacing the medium every 2 to 3 days.
8. After 1 week remove the 6 well plates containing the scaffolds to a laminar flood hood, remove the medium and invert each scaffold using sterile forceps, placing the mucosal surface uppermost. This time point is referred to as Day 0.
9. Place the steel rings on the mucosal surface in the center of the tissue as in step 5.4.
10. Prepare the T75 flasks of epithelial cells for trypsinization¹³. After the PBS wash and prior to the addition of trypsin-EDTA solution, add 2 ml of sterile 0.02% (w/v) EDTA solution to each flask. Incubate for 2 min at 37 °C. Tap the flask gently to selectively detach the i3T3 feeder layer while leaving the epithelial cells attached.
    Note: It is generally not practical or necessary to patient match the epithelial cells to the fibroblasts already incorporated in the model the previous week.
11. Pour off the EDTA solution containing the feeder layer and add trypsin-EDTA solution to harvest the epithelial cells¹³. Count the cells using a haemocytometer¹⁴. Centrifuge the cell suspension (10 min, 200 x g). Resuspend the cell pellet into an appropriate volume of Composite Medium 1 to produce a final cell count of 5 x 10⁶ cells per ml.
12. Add 0.2 ml of Composite Medium I, containing 1 x 10⁶ epithelial cells, within the ring. Flood the area around the outside of the ring with approximately 2 ml of Composite Medium I and place the constructs into the incubator at 37 °C in a 5% CO₂, humidified atmosphere.
13. After 24 hr (Day 1) remove the rings and medium and add at least 5 ml of fresh Composite Medium I, ensuring the scaffolds are fully submerged and return to the incubator. After a further 24 hr (Day 2) remove the medium and replaced with at least 5 ml of Composite Medium II, again ensuring the scaffolds are fully submerged and return to the incubator.
14. On Day 4 place sterile medical grade stainless steel mesh grids (2 cm wide x 2 cm long x 0.5 cm high), into fresh 6 well plates, one per construct. Use sterile tweezers to transfer the constructs onto the top of the steel grids, mucosal surface uppermost.
    1. Add sufficient Composite Medium III so that the liquid reaches the underside of the composite, but the surface is exposed to the air, this ensures that the sample is maintained at an air-liquid interface. The exact volume of medium required will vary depending upon the thickness of the scaffold, the volume of the well and the precise height of the stainless steel grid.
15. Maintain the composites at an air-liquid interface for between 10 and 20 days (Day 14 to Day 24), depending upon the requirements of the experiment. Replace the Composite Medium III with fresh medium every 2 to 3 days (Monday, Wednesday, Friday is a user-friendly regime).
    Note: After 5 days at an air-liquid interface (Day 9) the constructs have a sufficiently mature esophageal epithelium to be used in experiments of the impact of environmental factors on the esophageal epithelium.
16. At the end of the experiment, fix the constructs for histological analysis and immunohistochemical staining⁷, or extract protein¹⁵ or RNA^11,16 from the epithelium for further analysis. If required, retain the conditioned culture medium for analysis of extracellular signaling molecules¹⁷.

Results

This manuscript describes the process required, shown in schematic form in Figure 1, to culture 3D models of the human esophageal epithelium successfully. To confirm the suitability of the model as an experimental platform histological and immunohistochemical studies have been undertaken comparing the cultured tissues with normal human esophageal squamous mucosa.

Histological assessment of the epithelium produced by the method described shows a mature, multi-layered, stratifie...

Discussion

This manuscript describes the production and characterization of a biologically relevant human esophageal mucosal model suitable for use as an experimental platform to study the impact of exposure to environmental stressors upon the esophageal epithelium.

The most critical steps for the successful production of a human esophageal mucosal model are: ensuring that the majority of the epithelial cells remain proliferative and have not already begun to differentiate prior to seeding them on the sc...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Trypsin	BD Biosciences	215240	Prepare 0.1% w/v solution in PBS and filter sterilize. Warm in 37 °C water bath before use.
DMEM	Labtech	LM-D1112	Warm in 37 °C water bath before use
Ham's F12	Labtech	LM-H1236	Warm in 37 °C water bath before use
Foetal Calf Serum	Labtech	FB-1090
Epidermal Growth Factor	R+D Systems	236-EG-200	Prepare 200 µg/ml stock solution in 10 mM acetic acid, 1% FCS
Hydorcortisone	Sigma-Aldrich	H0396	Prepare stock solution in PBS and filter sterilize before use
Adenine	Sigma-Aldrich	A2786	Prepare stock solution in PBS and filter sterilize before use
Insulin	Sigma-Aldrich	I2767	Prepare 10 mg/ml solution in 0.01 M HCl, dilute 1:10 in distilled water and filter sterilize before use
Transferrin	Sigma-Aldrich	T2036	Prepare stock solution in distilled water and filter sterilize before use
Triiodothyronine	Sigma-Aldrich	T2752	Prepare stock solution in distilled water and filter sterilize before use
Cholera toxin	Sigma-Aldrich	C8052	Prepare stock solution in water
L-Glutamine	Sigma-Aldrich	G7513
Penicillin-Streptomycin	Sigma-Aldrich	P0781
Amphotericin B	Gibco	15290-026	Brand name Fungizone
PBS	Oxoid	BR0014	Dissolve 1 tablet in 100 ml water and autoclave to sterilize
Collagenase A	Roche	10103578001
Povidone-iodine solution	Ecolab	10830E	Brand name Videne
Ethanol	Sigma-Aldrich	E7023
Chelex 100	Sigma-Aldrich	C7901
Newborn calf serum	Gibco	26010074
Progesterone	Sigma-Aldrich	P8783	Prepare stock solution in DMEM and filter sterilize before use
Ethanolamine	Sigma-Aldrich	E9508	Prepare stock solution in DMEM and filter sterilize before use
Hydrocortisone	Sigma-Aldrich	H0888	Prepare stock solution in DMEM and filter sterilize before use use
O-phosphorylethanolamine	Sigma-Aldrich	P0503	Prepare stock solution in DMEM and filter sterilize before use
ITS (insulin, transferrin, selenium)	Lonza	17-838Z	Used for composite media preparation
Trypsin-EDTA	Sigma-Aldrich	T3924	Warm in 37 °C water bath before use
EDTA 0.02% solution	Sigma-Aldrich	E8008	Warm in 37 °C water bath before use
T75 culture flask	VWR	734-2313
50 ml centrifuge tube	Fisher	11819650
15 ml universal tube	SLS	SLS7504
180 ml pot	VWR	216-2603
Petri dish	SLS	150350
6 well plate	VWR	734-2323
stainless steel rings			Manufactured in house - medical grade stainless steel, internal diameter 10 mm, external diameter 20 mm
steel mesh grids			Manufactured in house - sheets have 0.3 cm diameter holes, bent to produce grid 2 cm (w) x 2 cm (d) x 0.5 cm (h)
ki67	Novocastra	KI67-MM1-L-CE	Clone MM1. Use at 1:100.
CK4	Abcam	ab9004	Clone 6B10. Use at 1:200.
CK14	Novocastra	LL002-L-CE	Clone LL002. Use at 1:200.
Involucrin	Novocastra	INV	Clone SY5. Use at 1:100.
OE21	Sigma-Aldrich	96062201
OE33	Sigma-Aldrich	96070808
Het-1A	ATCC-LGC	CRL-2692
Mouse 3T3 fibroblasts	ATCC-LGC	CRL-1658	previously growth arrested by irradiation (60 Gy)