Human embryonic lung organoid culture including recovering organoids from matrigel for immunostaining or RNA extraction

Embryonic lung organoid culture

1. Introduction

1. Tissue-derived organoids
2. Organoid culture
3. Genetic engineering of organoids
4. Conclusions

2. Protocols for experiment

2.1. Experimental plan

2.2. Splitting and maintenance of human embryonic lung organoids

2.3. Release of intact organoids from matrigel.

2.4. Isolation of single cells from human embryonic lung organoids.

3. Take home protocols

3.1. Organoid nucleic acid extraction.

3.2. Wholemount antibody staining of organoids.

4. Media protocols.

4.1. + Advanced DMEM/F12 +++

4.2. Self-renewing human embryonic lung organoid medium.

1. Introduction

1.1 Tissue-derived organoids

Adapted from Nikolic and Rawlins, 2017. (Nikolic and Rawlins, 2017) 

Traditionally cell culture has been carried out in two-dimensional (2D) model systems in which primary cells have to be transformed, for example by telomerase activation, in order for them to survive long-term in the in vitro environment. The process of transformation and long-term maintenance in 2D culture results in changes to the cells so that they no longer closely resemble their cell of origin. Such cell lines have been, and continue to be, extremely useful for studying fundamental cell biological mechanisms like cell division and DNA repair. However, they are extremely limited in their capacity to serve as models of tissue biology. By contrast, organoids are defined as three-dimensional (3D) structures derived from stem cells and consist of organ-specific cell types which self-organise through cell sorting and spatially restricted lineage commitment in a manner reminiscent of the native organ with some degree of organ functionality (Bredenoord et al., 2017; Huch and Koo, 2015). Organoids have also been referred to as “mini-organs” and enable in vitro modelling of organ development, disease modelling, including of viral infections and cancer, and drug screening (Dekkers et al., 2013; Drost and Clevers, 2018; Ramani et al., 2018). The 3D culture is thought to preserve native DNA integrity and prevents the cells from being transformed (Huch et al., 2015). Organoids were first successfully derived from mouse small intestine using single Lgr5⁺ stem cells (Sato et al., 2009). These organoids were entirely epithelial illustrating that organoids can be built without a non-epithelial cellular niche. The intestinal organoid is structured as crypt-villus units, with a similar stem cell hierarchy to in vivo, showing epithelial cell interactions are sufficient for the creation of crypt-villus units. Further work on cell-cell interactions using these intestinal organoids showed that essential niche signals are provided by Paneth cells, which are found interspersed between Lgr5⁺ stem cells (Sato et al., 2011b).

Organoids can be derived from tissue stem cells, either from the adult or the developing embryo. These usually consist of epithelial cells only, but co-cultures with other cell types can be performed to study signalling interactions (Lechner et al., 2017; Lee et al., 2014). Alternatively, organoids can be derived from pluripotent stem cells which have been differentiated towards a tissue-specific stem cell fate. In the latter case organoids usually include epithelial and non-epithelial cell lineages, although in general the non-epithelial cells are less well characterized (Aurora and Spence, 2016; Chen et al., 2017). Both approaches are equally valid and the most appropriate in any given situation will depend on the question being asked. For example, organoids derived from the endogenous organ have the advantage of initiating from a defined cell type. Moreover, they can usually be maintained in a long-term self-renewing state and, so far, most closely recapitulate the adult system. Although it is important to remember that tissue-derived organoids are in vitro models and do not perfectly recapitulate organ physiology. The organoid initiating cells from pluripotent cell cultures are often unknown and these organoids typically have a more embryonic-like character.  However, the pluripotent stem cell derived organoids have the advantage of an effectively unlimitless supply, even from humans, making them particularly attractive for large scale drug screening.

1.2 Organoid culture

Organoid growth requires the initiating stem cell population to both self-renew, to increase organoid size, and to differentiate. Organoids have been successfully cultured from multiple organs including, adult mouse stomach (Barker et al., 2010); mouse and human intestine/colon (Fujii et al., 2018; Jung et al., 2011; Sato et al., 2011a; Yin et al., 2014); mouse and human pancreas (Huch et al., 2013b; Loomans et al., 2018); mouse and human liver (Hu et al., 2018; Huch et al., 2013a); mouse and human prostate (Karthaus et al., 2014); mouse embryonic pancreas (Greggio et al., 2013) and human and mouse embryonic lung (Miller et al., 2018; Nichane et al., 2017; Nikolic et al., 2017). In most of these studies the same tissue culture medium supported both stem cell self-renewal and differentiation, for example intestinal stem cells self-organise efficiently into organoids and differentiate (Sato et al., 2009). By contrast, adult liver and pancreas organoids can be expanded, but do not differentiate easily yet (Huch et al., 2013a; Huch et al., 2013b). Similarly, mouse embryonic pancreas progenitors were expanded in a self-renewing medium and then switched to a differentiation medium for maturation (Greggio et al., 2013). This switch in medium composition may be particularly important for organoids derived from embryonic progenitors as such cells are typically reliant on extrinsic signalling from the adjacent mesenchyme in vivo (Laresgoiti et al., 2016; Rognoni et al., 2016). Cell-cell interactions within organoid cultures are likely to be just as important for differentiation as they are in vivo, through a process termed the “community effect”. This is now a well-established phenomenon in which the differentiation ability of a cell is enhanced by neighbouring cells differentiating in the same way simultaneously (Gurdon, 1988). 

The organoids have to be provided with an extracellular matrix to be able to initiate growth in vitro. Adult stem cells are typically seeded in matrigel, reconstituted extracellular matrix secreted by a tumour cell line (Schneeberger et al., 2017). It is widely-used and extremely convenient to grow cells in, although it suffers from batch-batch variation. One of the frontiers that is being explored for improving organoid culture is synthetic extracellular matrix, with one significant idea being that minimal cues should be provided for the stem cells to attach which could then secrete/remodel the matrix they need themselves. So far this idea has been successfully applied to intestinal organoid cultures which are by far the most well-developed organoid culture system (Gjorevski et al., 2016). 

1.3 Genetic engineering of organoids

If organoids are initiated from adult mice they can be grown from any available mouse strain which has been genetically-engineered in traditional fashion using embryonic stem cells. Similarly, organoids derived from pluripotent stem cells can be grown from genetically engineered pluripotent stem cells. By contrast, if organoids are grown from human tissue in particular, there can be a pressing need to perform genetic manipulation experiments for multiple purposes (Driehuis and Clevers, 2017). Retroviral, including lentiviral, piggybac and electroporation transduction protocols have all been published for organoid culture, although optimisation is required on a per cell type basis (Broutier et al., 2016; Fujii et al., 2015; Koo et al., 2011). Similarly, over expression experiments, as well as CRISPR-Cas9 knock-outs and gene-targeting have all been described (Drost et al., 2017; Matano et al., 2015), including phenotypic rescue of cystic fibrosis organoids by gene correction (Schwank et al., 2013). Optimisation of these techniques is on-going and the most-successful applications to date have used simple selection techniques, such as removal of specific growth factors from the culture medium.

1.4 Conclusions

Tissue-specific organoids are fast becoming a model of choice for their ability to recapitulate many aspects of organ physiology in vitro and their many, varied uses. The techniques for optimising organ cultures are developing rapidly. Genetic engineering techniques for application to organoid culture are developing at a similar pace and will a useful tool for developing improved human physiology and disease models.

2. Protocols for experiment

2.1. Experimental plan

In this practical we will learn/demonstrate how to passage expanding organoid cultures using human embryonic lung organoids as an exemplar system (Nikolic et al., 2017). In addition, we will practice isolating intact organoids from matrigel and preparing single cell suspensions to use for downstream genetic manipulation. A similar matrigel-removal technique can be used in preparation for wholemount antibody staining or nucleotide extraction (take home protocols). We will also visualize fluorescently-labelled organoids and discuss methods of genetic manipulation.

For passaging, we will estimate the confluency of our organoids and then manually remove them from the matrigel by washing in excess cold medium. We will practice breaking the organoids into smaller pieces by shear force using a P200 pipette. Organoid pieces will then be washed and resuspended at appropriate density in matrigel for replating. 

For preparation of a single cell suspension we will remove the organoids from the matrigel using Corning Cell Recovery Solution followed by enzymatic digestion with recombinant trypsin (TrypLE Express enzyme).
 

2.2. Splitting and maintenance of human embryonic lung organoids

Before you start

• Thaw matrigel on ice.
• Pre-warm plates by putting them into the incubator.
• Prepare sufficient quantities of ice-cold wash medium.

Things you will need

48-well tissue culture plates (Greiner Bio One cat.no. 677980)

15ml falcon tubes

Pipettes and filter tips (p1000, p200, p20)

Matrigel, Basement Membrane Matrix, Growth Factor Reduced (GFR), Phenol Red-free (BD cat.no. 356231)

Cold Advanced DMEM/F12 (ThermoFisher Scientific 12634010) for washing

Human embryonic lung self-renewal growth medium (protocol 4.2)

Splitting the organoids

1. Pipette 1ml cold Advanced DMEM/F12 into the well containing the organoids to break the basement membrane (matrigel) bubble.
2. Aspirate the matrigel and organoids into the 1ml pipette and transfer to a 15 ml falcon tube.
3. Add approximately 10 ml of cold Advanced DMEM/F12 into the falcon tube and invert 2-3 times.
   Washing in cold medium removes the old matrigel.
4. Spin 200-500 rcf, 5 minutes at room temperature. Aspirate media leaving about 1 ml.
5. Repeat the wash (steps 3 and 4) and place the tube containing organoids on ice.
6. Using a P200 pipette, manually break up the organoids by pipetting up and down approximately 20 times.
7. Add approximately 10 ml of cold Advanced DMEM/F12 into the falcon tube.
8. Spin 200-500 rcf, 5 minutes at room temperature, aspirate as much medium as possible and transfer the organoids to ice.
9. Resuspend the pellet in 30 – 150μl matrigel and pipette up and down to mix. 
   Typically we add 30μl matrigel per well of 48 well plate (ie resuspend the pellet in 150μl matrigel if splitting organoids at a 1:5 ratio).
10. Seed into a pre-warmed 48 well plate (30μl matrigel/organoid mix per well). Pipette slowly to avoid bubbles and seed organoids in the centre of the well to generate a bubble/dome of matrix.
11. Allow to solidify in the incubator for 10 – 15 minutes for the basement membrane mix to polymerize. 
12. Overlay with 300μl self-renewing medium per well. 

Note: organoids can also be grown in 24 well plates using 50μl matrigel per well and 600μl self-renewing medium.

For routine maintenance replace the culture medium every 3-4 days. 

2.3. Release of intact organoids from matrigel.

Before you start

• Prepare sufficient quantities of ice-cold wash medium.

Things you will need

15ml falcon tubes

Plastic Pasteur pipettes

Pipettes and filter tips (p1000, p200, p20)

Cold Advanced DMEM/F12 (ThermoFisher Scientific 12634010) for washing.

Cold Advanced DMEM/F12 (ThermoFisher Scientific 12634010) containing 10% FBS. 

Corning Cell Recovery Solution (Corning 354243)

Release of organoids from matrigel

1. Cut the end of a plastic Pasteur pipette to increase diameter.
2. Coat the Pasteur pipette with medium containing 10% FBS.
3. Add excess cold FBS-containing medium to the well containing the organoids.
4. Remove matrigel containing the organoids with the Pasteur pipette and transfer to a fresh falcon tube.
5. Add 10 ml cold Advanced DMEM/F12 (FBS is not required for this step).
6. Incubate on ice 10 min. Invert every ~2 mins. (This removes most of the matrigel).
7. Incubate on ice for 5 mins to allow the organoids to sediment.
8. Remove the medium (leave ~500 μl behind).
9. Repeat the washing steps (numbers 5-9) two more times until the matrigel is dissolved.
10. Add 3 ml Corning Cell Recovery Solution (Corning 354243) and incubate on ice 30 mins. Invert every 10 mins. 
11. Spin 200-500 rcf, 5 mins, 4°C.
12. Remove corning solution and wash in 10 ml cold Advanced DMEM/F12. pin 200-500 rcf, 5 mins, 4°C.
13. Transfer organoids to downstream protocol for use (e.g. single cell isolation, fixation for wholemount antibody staining, lysis). 
     

2.4. Isolation of single cells from human embryonic lung organoids.

Before you start

• Pre-warm TrypLE enzyme to 37°C.
   

Things you will need

Pipettes and filter tips (p1000, p200, p20)

15 ml Falcon Tubes

Advanced DMEM/F12 +++ (see media protocols) for washing.

TrypLE Express Enzyme 1X (ThermoFisher Scientific 12605010)

30 μm cell strainers (CellTrics® filters 30um, Sysmex, 04-004-2326)

Single Cell Dissociation

1. Start with isolated, intact organoids from protocol 2.3.
2. Remove the medium from the organoid culture. 
3. Wash the well with 1x PBS. 
4. Remove the PBS and add 500 μl of pre-warmed TrypLE express enzyme.
5. Triturate (pipette up and down 10 times) with P1000 pipette to break up the organoids and incubate at 37°C for 5 minutes.
6. Triturate by pipetting up and down with P200 pipette about 20 times.
7. Check that single cells have been obtained (if not, incubate a further 2-5 minutes at 37°C) and add 1 ml cold Advanced DMEM/F12 +++ to stop the reaction.
8. Filter through a 30 μm cell strainer and count cell number.

Note: if single cells are to be used to seed new organoid cultures, add 10 μM Rho Kinase inhibitor (Y27632) to the culture medium for the first 2 days.

3. Take home protocols

3.1. Organoid DNA or RNA extraction.

Before you start

• Prepare sufficient quantities of ice-cold wash medium.

Things you will need

15ml falcon tubes

Plastic Pasteur pipettes

Pipettes and filter tips (p1000, p200, p20)

Cold Advanced DMEM/F12 (ThermoFisher Scientific 12634010) for washing.

Cold Advanced DMEM/F12 (ThermoFisher Scientific 12634010) containing 10% FBS. 

Corning Cell Recovery Solution (Corning 354243)

RNA extraction kit, such as Qaigen RNAeasy Mini Kit, or TRIzol reagent (ThermoFisher Scientific, 15596026).

Or, DNA extraction kit, such as Qiagen QIAamp Fast DNA Tissue Kit (51404)

Organoid nucleic acid extraction

1. Cut the end of a plastic Pasteur pipette to increase diameter.
2. Coat the Pasteur pipette with medium containing 10% FBS.
3. Add excess cold FBS-containing medium to the well containing the organoids.
4. Remove matrigel containing the organoids with the Pasteur pipette and transfer to a fresh falcon tube.
5. Add 10 ml cold Advanced DMEM/F12 (FBS is not required here).
6. Incubate on ice 10 min. Invert every ~2 mins. (This removes most of the matrigel).
7. Incubate on ice for 5 mins to allow the organoids to sediment.
8. Remove the medium (leave ~500 μl behind).
9. Repeat the washing steps (numbers 5-9) two more times until the matrigel is dissolved.
10. Add 3 ml Corning Cell Recovery Solution (Corning 354243) and incubate on ice 30 mins. Invert every 10 mins. 
11. Spin 200 rcf, 5 mins, 4°C.
12. Remove corning solution and wash in 10 ml PBS. Spin 200 r.c.f.  5 mins, 4°C.
13. Resuspend in lysis buffer for DNA or RNA extraction and follow downstream protocol.

3.2. Wholemount antibody staining of organoids.

Before you start

• Prepare sufficient quantities of ice-cold wash medium.
• Prepare fresh 4% PFA.

Things you will need

15ml falcon tubes

Plastic Pasteur pipettes

Pipettes and filter tips (p1000, p200, p20)

Cold Advanced DMEM/F12 (ThermoFisher Scientific 12634010) for washing.

Cold Advanced DMEM/F12 (ThermoFisher Scientific 12634010) containing 10% FBS. 

Corning Cell Recovery Solution (Corning 354243)

Freshly made 4% paraformaldehyde on ice.

PBS

Wash buffer: PBS containing 0.5% BSA and 0.2% Triton X-100

Blocking buffer: PBS containing 1% BSA, 5% normal donkey serum and 0.2% Triton X-100

Appropriate primary and secondary antibodies

Mounting medium e.g. Vectashield (Vector Laboratories H-1000)

Slides

Coverslips

Grace Bio-labs Secure-seal Imaging spacers 20 mm Diameter x 0.12 mm Depth 654006 (Sigma-Aldrich GBL654006)

Wholemount antibody staining of organoids

1. Cut the end of a plastic Pasteur pipette to increase diameter.
2. Coat the Pasteur pipette with medium containing 10% FBS.
3. Add excess cold FBS-containing medium to the well containing the organoids.
4. Remove matrigel containing the organoids with the Pasteur pipette and transfer to a fresh falcon tube.
5. Add 10 ml cold Advanced DMEM/F12 (FBS is not required here).
6. Incubate on ice 10 min. Invert every ~2 mins. (This removes most of the matrigel).
7. Incubate on ice for 5 mins to allow the organoids to sediment.
8. Remove the medium (leave ~500 μl behind).
9. Repeat the washing steps (numbers 5-9) two more times until the matrigel is dissolved.
10. Add 3 ml Corning Cell Recovery Solution (Corning 354243) and incubate on ice 30 mins. Invert every 10 mins. 
11. Spin 200 r.c.f.  5 mins, 4°C.
12. Remove corning solution and wash in 10 ml PBS. Spin 200 r.c.f.  5 mins, 4°C.
13. Remove PBS and add 3 ml 4% PFA and incubate 30 mins on ice.
14. Discard PFA by local approved disposal route.
15. Add 10 ml wash buffer and spin 200 rcf, 5 mins, 4°C.
16. Wash again in 10 ml wash buffer
17. If necessary store at 4°C in wash buffer up to 72 hours before beginning wholemount staining. 
18. Transfer organoids to a conical-bottomed 96 well plate to begin wholemount antibody staining protocol using a cut P1000 tip which is coated in wash buffer. 
    All subsequent wash steps are performed on a rotating platform and wash medium is removed from the organoids slowly under a dissecting microscope to avoid aspiration of organoids.
19. Perform 3x 5 minute washes at room temperature in wash buffer.
    Do not try to remove all the buffer after each wash to avoid aspirating the organoids.
20. Add blocking buffer and incubate at 1 hour at room temperature.
    Can incubate in block at 4°C overnight.
21. Replace the block with primary antibodies diluted to appropriate concentrations in blocking buffer. Incubate overnight at 4°C.
    Ensure the organoids are fully covered. 100-200 μl primary antibody mix is usually sufficient.
22. The next morning wash 3x briefly in wash buffer.
23. Wash 3x 10-20 minutes at room temperature in wash buffer.
24.  Replace wash buffer with secondary antibodies diluted to appropriate concentration in PBS containing 0.2% triton and 5% normal donkey serum. Incubate room temperature for 2-3 hours.
    Can incubate in secondary antibodies at 4°C overnight if convenient.
25. Wash 3x briefly in wash buffer.
26. Wash 3x 10-20 minutes at room temperature in wash buffer.
27. Wash in wash buffer containing DAPI for 30 minutes at room temperature. 
28. Wash 3x briefly in wash buffer.
29. Wash 3x 10-20 minutes at room temperature in wash buffer.
30. Attach a Grace BioLabs imaging spacer to the centre of a microscope slide.
31. Using a cut P200 tip slowly suck up the organoids and allow them to drop to the end of the tip. Mount organoids in the centre of the imaging spacer in a small volume of wash buffer. Remove excess wash buffer with a P20 tip. 
32. Gently pipette 50 μl of fluorescent mounting medium on top of the organoids and mount a coverslip on top of the imaging spacer.

33. Seal with nail polish and store at 4°C until imaging.

     

4. Media protocols

4.1 Advanced DMEM/F12 +++ medium

4.2 Human embryonic lung self-renewing medium, 20ml

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