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.
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.
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 proliferation2-4.
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 vectors6,7. 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.
1. Preparation of Polyethylenimine (PEI) as Transfection Reagent
2. Production of Lentiviral Particles
Day 1:
Day 2:
Day 4:
Day 5:
3. Lentiviral Transduction of Organoids
Day 0:
Day 2:
Day 5:
Day 7:
4. Organoid RNA Preparation for Quantitative RT-PCR or Microarray
5. Processing Organoids for Paraffin Embedding and Immunohistochemistry
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 were trypsinized to allow penetration of virus particles to single cells. When transducing cells with lentiviral particles, a number of methods may be tried to enhance transduction efficacy such as spinoculation or prolonged incubation. High titer PGK-eGFP lentivirus was used to enable visualization of transduction efficacy by fluorescent microscopy. Transduction efficacy of organoids with this plasmid was high and approached 100% (Figure 4E, F). Improving efficacy using spinoculation (Figure 4A, B) or prolonged incubation with lentiviral particles (Figure 4C, D) did therefore not yield additional value.
RNA extraction
Next organoids for RNA extraction were grown. Full-grown organoids were harvested that were subjected to gamma irradiation or control treatment to show reduced RNA integrity (Figure 5). Upon irradiation with 6 Gy, RNA is degraded and compared to control treated organoids, the RNA integrity number (RIN) is reduced.
Immunohistochemistry on paraffin embedded organoids
After incubating organoids for 2 hours in culture medium supplemented with BrdU, organoids in formalin were fixed and processed for immunohistochemistry (Figure 6). Using mouse anti BrdU, proliferative cells could be observed in crypt-segments of organoids and not in the differentiated compartment.
Figure 1: Schematic of lentivirus production for organoid transduction. As written in protocol part 2, in this schematic, the most critical steps of virus production are represented with protocol step numbers and timing.
Figure 2: Schematic of lentiviral transduction of organoids. As written in protocol part 3, in this schematic, the most critical steps of organoid transduction are represented with protocol step numbers and timing.
Figure 3: Organoids prior to and during transduction. Normally growing organoids (A) are split into densely growing small organoids (B) that become cystic after incubation with Chir99021 for a number of days (C). Upon dissociation of these organoids using trypsin, single cells and small clumps of cells remain (D) that are transduced subsequently. Scale bar 100 µm.
Figure 4: Transduction of organoids using lentiviral expression vectors. Brightfield images (A, C, E, G) and fluorescent images (B, D, F, H) from organoids that were transfected with either PGK-eGFP lentivirus (A–F) or control lentivirus (G, H). eGFP expression in organoids after lentiviral transduction using both spinoculation and extended incubation (A, B), spinoculation only (C, D) or no additional steps to increase transduction efficacy (E, F), compared to control vector transduced organoids, that do not express eGFP (G, H). Note that using PGK-eGFP lentiviral construct, transduction efficacy is not greatly increased by additional steps. Scale bar 100 µm.
Figure 5: Representative result of RNA preps from organoids on bioanalyzer. Note strong demarcation of bands representing high RNA integrity in lanes 2-4 and smear around bands on lanes 5-7 representing log RNA integrity. RNA integrity number (RIN) of bands 2-4 is 8.7, 9.2 and 9 respectively, whereas the RIN of bands 5-7 is 6.4, 6.6 and 5.5.
Figure 6: Immunohistochemical analysis of formalin fixed paraffin embedded organoids. Formalin fixed paraffin embedded 4 µm section of organoids cultured in the presence of BrdU for two hours and fixed subsequently. Section is stained with anti-BrdU antibody. Scale bar 100 µm. Please click here to view a larger version of this figure.
Basic medium | Additions | |
Cell line culture medium | DMEM | 10% FCS |
1% penicillin/streptomycin | ||
2 mM Glutamine | ||
Organoid culture medium | Advanced DMEM F12 | HEPES |
1% penicillin/streptomycin | ||
1x glutamax | ||
1% N2 supplement | ||
2% B27 supplement | ||
125 nM n-acetyl cysteine | ||
mouse Egf (50 ng/ml) | ||
10% Nog-Fc conditioned medium (equivalent to 100 ng/ml) | ||
10% Rspo1-Fc conditioned medium (equivalent to 500 ng/ml) |
Table 1: Culture medium composition.
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 Chir99021 increases proliferation and thereby viability after transduction. It is important to maintain small cell clusters after trypsinization, since single cells have decreased survival. In contrast to viral transductions using murine retrovirus, lentiviral transductions are more efficient and ordinarily do not require spinoculation, a technique known to increases transduction efficacy in ES cells10. When using murine retroviruses or when lentiviral transduction yields low efficacy, spinoculation can optionally be utilized to increase transduction rate. We do not regularly assess virus titer, since all available virus from a single round of production for transduction of organoids derived from maximally two 0.95 cm2 wells was used. In addition, generally use antibiotic selection for removal of non-transduced cells. Assessment of viral titer may however be of value when encountering problems with transduction efficacy.
Identical to adherent cells, the level of selection antibiotic can increase the number of viral integrations and thereby expression of the transgene. Use high-level puromycin (10 µg/ml) to obtain high transgenic expression and effective knockdown, but when organoids exhibit signs of toxicity, lower levels may be used (minimally 1 µg/ml). In addition, alternative selection antibiotics may be used or selection may be omitted, although transduction is likely to be incomplete and this may not result in long-term stable expression. Furthermore, since lentiviral integration in the genome is heterogeneous throughout a population of cells, expression of the transgene may be subdue to changes after culturing cells for a prolonged time. This may result from selective pressure by expression of the transgene. Although antibiotic selection limits these changes, they may remain, especially in the case of constitutive expression of transgenes. This disadvantage may be overcome by single cell cloning of organoids, but this technique is time consuming. Lentiviral transduction can be performed with a wide variety of plasmids that result in either stable or inducible expression of transgenes. These transgenes are usually expressed from ubiquitous promoters, such as the CMV or the PGK promoter and may result in supernatural expression. Similar to transgenic overexpression in cancer cell lines or model animals, caution is warranted interpreting results from overexpression.
For experiments that require RNA, the low number of viable cells normally grown in a single 0.95 cm2 well (standard surface area of 48 well) in contrast to adherent cells that can easily be grown in large flasks is the rate-limiting factor for quality and multitude of downstream analysis. We find that normally, a single well of approximately 50 full grown organoids yields between 0.4 μg and 1 μg total RNA, being sufficient for quantitative RT-PCR and RNA microarray without further amplification. Certain treatment regimens or genetic alterations can reduce the RNA content significantly however. A first step to increase total RNA is pooling of multiple wells.
For paraffin embedding of organoids, it is critical to obtain sufficient material in order to visualize organoids during the process. Addition of minute amounts of eosin to fixed organoids allows visualization of organoids during the whole process, but is not required for normal embedding and may be omitted when this step interferes with further analysis. Usually, paraffin-embedding cassettes have holes in them to allow flow of fixative and process solutions to the tissue in the cassette.
Lentiviral organoid transduction and preparation for downstream standard techniques increases scientific potential of these cultures and raises the standard in cell culture to three-dimensional techniques from primary intestinal epithelium. The range of downstream experiments to be performed is however not limited to techniques described in the current protocol and may encompass all techniques performed on cell lines or mice, given the amount of cellular material generated by culture is sufficient. In research on specific genetic elements and their function in the intestinal epithelium, homologous recombination techniques in model animals, most notably mice, remain the gold standard. Cultures of organoids do not contain mesenchymal or immunological niche cells and cultured crypts are ever expanding, contrasting the situation found in vivo. Nonetheless, these cultures have raised the quality of in vitro findings greatly and taking into account the myriad downstream techniques that can be performed, organoid culture has and will greatly enhance research on the intestinal epithelium.
The authors have nothing to disclose.
J. Heijmans is supported by a stipendium from the Dutch cancer foundation (KFW). G.R. van den Brink is supported by funding from the European Research Council under the European Community’s Seventh Framework Program (FP7/2007-2013)/European Research Council grant agreement number 241344 and by a VIDI grant from the Netherlands Organization for Scientific Research (GvdB).
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 |