Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Summary

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Abstract

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

Introduction

Over the past decade, the field of cancer research has demonstrated renewed enthusiasm for patient-derived xenografts (PDXs) as a tool for assessing cancer cell pathway reliance and drug susceptibility¹. The most common PDX models are established by subcutaneous or orthotopic implantation of human tumor cells—a tumor fragment, a cluster of dissociated tumor-derived cells, or a sample of isolated circulating tumor cells (CTCs)—into a rodent host. If the tumor “take” is successful, the xenograft cells will proliferate, vascularize, and otherwise interact with the host tissue to create a tumor, which can be harvested at an ....

Protocol

Tumor tissue was obtained with patient consent and according to an approved Institutional Review Board (IRB) protocol. Xenografts were implanted, grown, and harvested according to an accepted Institutional Animal Care and Use Committee (IACUC) protocol.

NOTE: All work is to be performed in a sterile biological safety cabinet to maintain sterility. All steps should be conducted at room temperature unless otherwise specified.

1. Preparation of materials for PDX processing

1. Autoclave forceps and scalpel handle or razor blades.
2. Thaw dissociation enzyme solution at 4 °C overnight or at room t....

Results

A programmable perfusion rocker was prepared in a standard water-jacked cell culture incubator, and two-lane microfluidic plates were prepared in a standard biosafety cabinet for loading (Figure 1). An MDA-PCA-118b PDX tumor was expanded in vivo, harvested when it had reached a maximum size, and dissociated as described in protocol section 2 to create a slurry suspension of cells, at approximately a single-cell state (Figure 2A). The slurry was dispensed into 6-.......

Discussion

Here we describe a method for processing and culturing viable PDX-derived tumor cells in a high-throughput, perfused 3D culture system. While this protocol utilizes PCa PDX tissue, it is equally effective for other epithelial-derived tumors. Tumor characteristics vary between individual PDX lines even within the same tissue of origin (prostate, breast, etc.). Some PCa PDX lines are more fibrotic and difficult to isolate viable cells from while others are more cellular. The tumor size noted here can be varied within IACUC.......

Materials

Name	Company	Catalog Number	Comments
1N NaOH			any suitable tissue culture grade
60 mm round tissue culture dishes			any suitable
6-well tissue culture plates			any suitable
70 µm cell strainers	Corning	431751	or equivalent
Centrifuge	Eppendorf	5810R with suitable rotor and buckets for 15/50 mL conical centrifuge tubes	or equivalent
Density gradient centrifugation solution	Millipore Sigma	P1644	Percoll
Dimethylsulfoxide			any suitable tissue culture grade
Dissociation enzyme solution	StemCell Technologies	07921	ACCUMAX
DMEM-F12	ThermoFisher Scientific	11039021	or equivalent
Forceps			any suitable
HA hydrogel kit	ESI BIO	GS311	HyStem (Hyaluronic acid-SH and PEGDA)
Hanks Balanced Salt Solution	Lonza	10-527F	or equivalent
Heat-inactivated fetal bovine serum	Atlanta Biologicals	S11150
Hemocytometer	Fisher Scientific	02-671-51B Hausser BrightLine	or equivalent
Hoechst 33342	ThermoFisher Scientific	H1398	or equivalent
Image processing software	Oxford Instruments	Imaris 9.3	or equivalent
LIVE/DEAD Cell Viability/Cytotoxicity Kit (Calcein-AM/Ethidium Homodimer-1)	ThermoFisher Scientific	L3224	or equivalent
Microfluidic culture plate	Mimetas	9603-400-B	2-lane OrganoPlate
Microscope	Nikon	A1R	or equivalent
Multichannel pipette	Eppendorf	3125000036	or equivalent
PDX-derived tumor tissue			obtained under IRB approval for human tissue and IACUC approval for animal host
Penicillin-streptomycin	ThermoFisher Scientific	15140-122	or equivalent
Perfusion rocker	Mimetas	OrganoPlate Perfusion Rocker Mini
pH strips (pH 5-9)			any suitable
Phosphate-buffered saline solution	Lonza	17-516F	or equivalent
Razor blades			any suitable
Rotating xy-shaker	VWR	Advanced 3500 Orbital Shaker	or equivalent
Scalpel handle			any suitable
Single channel repeating pipette	Eppendorf	22260201
Sterile, 15mL conical centrifuge tubes			any suitable
Sterile, 50mL conical centrifuge tubes			any suitable