Een protocol voor Lentiviral Transduction en Downstream Analyse van Intestinale Organoids

Summary

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Abstract

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

Introduction

The intestinal epithelium is one of the most rapidly proliferating bodily tissues, which has caused it to attract wide interest from research on cancer and stem cells. In 2009 a technique was published to generate long lasting cultures of small intestinal crypts in matrigel, conserving a 3 dimensional structure¹. These structures, termed intestinal organoids, can be cultured using standard techniques, with surrounding medium supplemented with a number of defined growth factors, including the Bmp-signaling pathway inhibitor noggin (Nog), the Wnt-signaling pathway enhancer rspondin 1 (Rspo1) and epidermal growth factor (Egf) all found to enhance intestinal proliferation^2-4.

Organoids surpass traditional cancer cell lines in the aspects that they are non-mutated, have maintained stem cell hierarchy, display intact cellular polarization and exhibit differentiation into all cell lineages found in the nascent small intestinal epithelium. Since they can be transduced to carry transgenes or RNA interference constructs⁵, they are used to study specific genetic elements, outweighing experiments using transgenic mice in facets of cost and speed. Transgenic expression in organoids can be performed using either murine retroviral or lentiviral vectors^6,7. Due to the limitations of murine retroviruses, capable of transducing mitotic cells exclusively⁸, lentiviral transduction is more frequently used for cells that are difficult to infect, such as organoids.

Virally transduced and stably expressing transgenic organoids can be used for a multitude of downstream analyses, including quantitative RNA analyses and immunohistochemistry. Taken together, culture of organoids from primary intestinal epithelial cells has evolved into a routine technique that is easy to implement without specific laboratory requirements, and has become the novel standard in cell culture in research on the intestinal epithelium.

Techniques of viral transduction and subsequent downstream analysis in organoids are tedious to perform and to aid organoid experiments we generated this video protocol, showing methods for lentiviral transduction of cultured organoids. We additionally show how correct processing of organoids can increase yield and therefore enhance performance of downstream analysis using RNA techniques or immunohistochemistry. In the protocol, organoids that are derived from small intestinal crypts were exclusively used, although the techniques described may be applied to colonic organoids as well.

Protocol

1. Preparation of Polyethylenimine (PEI) as Transfection Reagent Dissolve approximately 150 mg of PEI into 100 ml of H2O. Adjust solution to pH 7.4 by adding HCl until solution becomes clear and stir until completely dissolved. This may take between 10 and 60 min and add water to an end concentration of 1 mg/ml. When clear, filter the PEI solution through sterile 0.22 µm filter and store in a -80 °C freezer in aliquots of 5 ml. 2. Produ…

Representative Results

Organoid lentiviral transduction The technique of organoid transduction using lentiviral particles depends on correct handling of organoids prior and during transduction. Organoids (Figure 3A) were cultured and they were disrupted into single crypts (Figure 3B). As previously reported, these single crypts, when cultured in the presence of the GSK3 inhibitor Chir99021 became cystic crypts9 (Figure 3C). Subsequently organoids…

Discussion

The current video protocol describes lentiviral transduction of organoids from primary intestinal epithelium and downstream analysis of these organoids using quantitative RNA techniques and immunohistochemistry.

Lentiviral transduction is often performed in adherent or floating cells in culture plates. Since the three-dimensional structure of organoids renders them difficult to penetrate by viral particles, a number of methods to increase efficacy are used. Pretreatment of organoids using Chir…

Materials

Reagent	Company	Cat. No.
Polyethylene imine	polysciences	23966-2
DMEM medium	Lonza	BE12-614F
Fetal calf serum	Lonza	DE14-801F
Penicillin-streptomycin	Invitrogen	15140-122
Glutamin	Invitrogen	25030-024
matrigel	BD	BD 356231
Advanced DMEM-F12	Gibco	12634-010
N2	Invitrogen	17502-048
B27	Invitrogen	17504-044
N-acetyl cysteine	Sigma	A9165-1G
mouse Egf	Invitrogen	PMG8045
Hepes 1M	Invitrogen	15630-056
glutamax 100x	Invitrogen	35050-038
Chir 99021	axon	1386
Y27632	Sigma	Y0503-5MG
polybrene	Sigma	107689
nicotinamide	Sigma	N0636
Trypsin	Lonza	BE02-007E
puromycin	sigma	P 7255
Rneasy mini kit	Qiagen	74106
b-mercaptoethanol	Merck	8,057,400,250
Ovation Pico WTA system	NuGen	3300-12
paraformaldehyde	Sigma	252549-1L
glass vial conical 12mm x 75mm 5ml	VWR	LSUKM12
Eosin Yellowish	VWR	1,159,350,025