The pituitary gland is the key regulator of the body’s endocrine system. This article describes the development of organoids from the mouse pituitary as a novel 3D in vitro model to study the gland’s stem cell population of which the biology and function remain poorly understood.
The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, and stress response. More than a decade ago, stem cells were identified in the pituitary gland. However, despite the application of transgenic in vivo approaches, their phenotype, biology, and role remain unclear. To tackle this enigma, a new and innovative organoid in vitro model is developed to deeply unravel pituitary stem cell biology. Organoids represent 3D cell structures that, under defined culture conditions, self-develop from a tissue’s (epithelial) stem cells and recapitulate multiple hallmarks of those stem cells and their tissue. It is shown here that mouse pituitary-derived organoids develop from the gland’s stem cells and faithfully recapitulate their in vivo phenotypic and functional characteristics. Among others, they reproduce the activation state of the stem cells as in vivo occurring in response to transgenically inflicted local damage. The organoids are long-term expandable while robustly retaining their stemness phenotype. The new research model is highly valuable to decipher the stem cells’ phenotype and behavior during key conditions of pituitary remodeling, ranging from neonatal maturation to aging-associated fading, and from healthy to diseased glands. Here, a detailed protocol is presented to establish mouse pituitary-derived organoids, which provide a powerful tool to dive into the yet enigmatic world of pituitary stem cells.
The pituitary is a tiny endocrine gland located at the base of the brain, where it is connected to the hypothalamus. The gland integrates peripheral and central (hypothalamic) inputs to generate a tuned and coordinated hormone release, thereby regulating downstream target endocrine organs (such as adrenal glands and gonads) for producing appropriate hormones at the proper time. The pituitary is the key regulator of the endocrine system and is therefore rightfully termed the master gland1.
The mouse pituitary consists of three lobes (Figure 1), i.e., the anterior lobe (AL), the intermediate lobe (IL), and the posterior lobe (PL). The major endocrine AL contains five hormonal cell types, including somatotropes that produce growth hormone (GH); lactotropes generating prolactin (PRL); corticotropes that secrete adrenocorticotropic hormone (ACTH); thyrotropes responsible for thyroid-stimulating hormone (TSH) production; and gonadotropes that make luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The PL consists of axonal projections from the hypothalamus in which the hormones oxytocin and vasopressin (antidiuretic hormone) are stored. The IL is located in-between the AL and PL and houses melanotropes that produce melanocyte-stimulating hormone (MSH). In the human pituitary, the IL regresses during development, and melanotropes are spread within the AL1. In addition to the endocrine cells, the pituitary gland also contains a pool of stem cells, essentially marked by the transcription factor SOX22,3,4,5,6. These SOX2+ cells are located in the marginal zone (MZ), the epithelial lining of the cleft (an embryonic remnant lumen between the AL and IL), or are spread as clusters throughout the parenchyma of the AL, thereby proposing two stem cell niches in the gland (Figure 1)2,3,4,5,6.
Given the indispensable nature of the pituitary, malfunctioning of the gland is associated with serious morbidity. Hyperpituitarism (characterized by over-secretion of one or more hormones) and hypopituitarism (defective or missing production of one or more hormones) can be caused by pituitary neuroendocrine tumors (PitNETs; e.g., ACTH-producing tumors leading to Cushing's disease) or by genetic defects (e.g., GH deficiency resulting in dwarfism)7. In addition, pituitary surgery (e.g., to remove tumors), infections (e.g., hypothalamic-pituitary tuberculosis, or infections following bacterial meningitis or encephalitis), Sheehan's syndrome (necrosis because of insufficient blood flow due to heavy blood loss at birth-giving), pituitary apoplexy and traumatic brain injury are other important causes of pituitary hypofunction8. It has been shown that the mouse pituitary possesses the regenerative capacity, being able to repair local damage introduced by transgenic ablation of endocrine cells9,10. The SOX2+ stem cells acutely react to the inflicted injury showing an activated phenotype, marked by enhanced proliferation (resulting in stem cell expansion) and increased expression of stemness-related factors and pathways (e.g., WNT/NOTCH). Moreover, the stem cells start to express the ablated hormone, finally resulting in substantial restoration of the depleted cell population over the following (5 to 6) months9,10. Also, during the neonatal maturation phase of the gland (the first 3 weeks after birth), the pituitary stem cells are thriving in an activated state6,11,12,13, whereas organismal aging is associated with declined in situ stem cell functionality, due to an increasing inflammatory (micro-) environment at aging (or 'inflammaging')10,14. In addition, tumorigenesis in the gland is also associated with stem cell activation7,15. Although stem cell activation has been detected in several situations of pituitary remodeling (reviewed in7,16), underlying mechanisms remain unclear. Since in vivo approaches (such as lineage tracing in transgenic mice) have not delivered a clear or comprehensive picture of pituitary stem cells, the development of reliable in vitro models to explore stem cell biology in normal and diseased pituitary is essential. Standard in vitro culture of primary pituitary stem cells remains inadequate because of very limited growth capacity and non-physiological (2D) conditions with rapid loss of phenotype (for a more detailed overview, see16). 3D sphere cultures (pituispheres) have been established from pituitary stem cells as identified by side population and SOX2+ phenotype2,3,4. The pituispheres clonally grow from the stem cells, express stemness markers and show differentiation capacity into the endocrine cell types. However, they do not considerably expand while showing only limited passageability (2-3 passages)3,4. Sphere-like structures were also obtained from non-dissociated pituitary stem cell clusters when cultured in 50% diluted Matrigel for 1 week, but expandability was not shown17. The pituisphere approach is mostly used as a read-out tool for stem cell numbers, but further applications are limited by inferior expansion capacity16.
To address and overcome these shortcomings, a new 3D model has recently been established, i.e., organoids, starting from the major endocrine AL of mice containing the MZ and parenchymal stem cells. It has been shown that the organoids are indeed derived from the pituitary's stem cells and faithfully recapitulate their phenotype18. Moreover, the organoids are long-term expandable, while robustly maintaining their stemness nature. Therefore, they provide a reliable method to expand primary pituitary stem cells for profound exploration. Such exploration is not achievable with the limited number of stem cells that can be isolated from a pituitary, which are also not expandable in 2D conditions16. It has been shown that the organoids are valuable and reliable tools to uncover new pituitary stem cell features (translatable to in vivo)14,18. Importantly, the organoid model faithfully mirrors the pituitary stem cell activation status as occurring during local tissue damage and neonatal maturation, showing enhanced formation efficiency and replicating upregulated molecular pathways14,18. Hence, the pituitary-derived organoid model is an innovative and powerful pituitary stem cell biology research model as well as a stem cell activation readout tool.
This protocol describes in detail the establishment of mouse pituitary-derived organoids. To this aim, the AL is isolated and dissociated into single cells, which are embedded in extracellular matrix-mimicking Matrigel (hereon referred to as ECM). The cell-ECM assembly is then cultured in a defined medium, essentially containing stem cell growth factors and pituitary embryonic regulators (further referred to as 'pituitary organoid medium' (PitOM)18; Table 1). Once the organoids are fully developed (after 10-14 days), they can be further expanded trough sequential passaging and subjected to extensive downstream exploration (e.g., immunofluorescence, RT-qPCR, and bulk or single-cell transcriptomics; Figure 1). In the longer run, it is expected that the pituitary stem cell organoids will pave the way to tissue repair approaches and regenerative medicine.
Animal experiments for this study were approved by the KU Leuven Ethical Committee for Animal Experimentation (P153/2018). All mice were housed at the university's animal facility under standardized conditions (constant temperature of 23 ± 1.5 °C, relative humidity 40%-60%, and a day/night cycle of 12 h), with access to water and food ad libitum.
1. Mice
2. Isolation and dissociation of mouse AL
NOTE: Medium A, B, and C are prepared in advance19,20. Compositions are shown in Table 2.
3. Establishment and culturing of AL-derived organoids
NOTE: Thaw ECM on ice in advance (2-3 h for 1 mL) and keep it on ice for the duration of the protocol.
4. Cryopreservation of AL-derived organoids and thawing
5. Validation of AL-derived organoids
After isolation and dissociation of the AL, the obtained single cells are seeded in ECM and grown in PitOM (Figure 1, Table 1). Figure 3A displays the cell culture and density at seeding (Day 0). Some small debris may be present (Figure 3A, white arrowheads), but will disappear at passaging. Fourteen days after seeding, the AL-derived organoids are fully developed (Figure 3A). The organoids exhibit a cystic morphology, with an epithelial layer that encloses a lumen. At this stage, the organoids reach a diameter of 500 µm and have to be passaged. Figure 3B shows the AL-derived organoid culture after passaging at the indicated time following re-seeding of the dissociated organoid fragments.
Occasionally, one or more dense structures may appear in the organoid culture (Figure 3A, Unfavorable). When passaging, dense organoids tend to take over, ending up in cultures with only dense structures after a couple of passages (Figure 3B, Unfavorable). Therefore, it is recommended not to proceed with wells that contain dense organoids (passage 0). Alternatively, dense organoids can be discarded by sedimentation, which leaves the cystic organoids to continue with. The origin of these dense organoids is at present unclear, but they show a less pronounced pituitary nature18. If organoids do not, or less efficiently regrow after passaging, dissociation procedures need to be optimized. In particular, one must pay attention not to dissociate too harsh; the organoids must be split up to fragments, not to single cells (Figure 3B, Day 0, inset).
Immunofluorescence staining analysis confirms the epithelial character of the AL-derived organoids, as they express the epithelial markers E-cadherin (E-Cad) and cytokeratin 8/18 (CK8/18; Figure 3C), which, moreover, have been described as stem cell markers in the pituitary18. The stemness nature of the organoids is additionally demonstrated by SOX2 and TROP2 expression, both of which were also identified as pituitary stem cell markers (Figure 3C)14,18. LHX3, a transcription factor specifically expressed in the (early-developing) pituitary, validates the organoids' pituitary phenotype (Figure 3C). Some of the organoid-constituting cells are in a proliferative state, expressing the proliferation marker Ki67 (Figure 3C).
Further exploration and validation of the pituitary (stemness) phenotype of the AL-derived organoids is performed with reverse transcription-quantitative PCR (RT-qPCR). High expression of the stemness markers Sox2, Cdh1 (encoding E-Cad), Krt8, Krt18 and Trop2 is present in the organoids, clearly higher than in primary AL, indicating that the organoids enrich for the stem cells and thus represent the AL stem cell compartment, as previously described (Figure 3D)18. Notably, the developmental transcription factors Pitx1 and Pitx2 remain expressed after development in several hormonal cell types in the AL, and hence their high expression in the AL as well. The cultures robustly retain their stemness phenotype, as demonstrated by the sustained (high) expression of these markers after multiple passages (Figure 3D).
Figure 1: Overview of the establishment, maintenance, characterization, and application potential of organoids from healthy and diseased pituitary. AL, anterior lobe; IL, intermediate lobe; PL, posterior lobe; MZ, marginal zone; PitOM, pituitary organoid medium (created with BioRender.com). Stem cell niches in the AL are indicated in purple. Please click here to view a larger version of this figure.
Figure 2: Isolation of the pituitary gland from adult euthanized mouse. Representative images consecutively taken following (A) decapitation, (B) removal of head skin (nose bridge is encircled), (C) opening of the cranium, and (D) removal of the brain, exposing the pituitary gland (encircled). Arrow points to the PL, which is discarded (together with the associated IL), leaving the AL for isolation and dissociation. Please click here to view a larger version of this figure.
Figure 3: Establishment and validation of AL-derived organoids. (A) AL cell seeding and organoid development in PitOM at indicated days (passage 0). The top row shows favorable organoid growth, with only cystic structures developing. The bottom row shows unfavorable growth with a large dense structure appearing (boxed). White arrowheads indicate debris, black arrowheads indicate single cells (magnified in inset). (B) Organoid fragments (magnified in inset) seeded at passaging (Day 0) and regrowth of organoids as observed 7 days later. The top row shows favorable organoid regrowth, with only cystic structures growing. The bottom row shows unfavorable regrowth with dense organoids taking over the culture. (C) Immunofluorescence staining of E-Cad, SOX2, TROP2 (all red), CK8/18, LHX3 and Ki67 (all green) in AL-derived organoids. Nuclei are labeled with Hoechst33342 (blue). Arrowheads indicate Ki67+ cells. Scale bars are indicated. (D) Gene expression analysis of stemness markers (Sox2, Cdh1, Krt8, Krt18, Trop2), and developmental transcription factors (Pitx1, Pitx2) in primary AL and AL-derived organoids (Passage 0 means 14 days after cell seeding) determined by RT-qPCR (mean ± SEM). Data points represent biological replicates. Delta cycle threshold (dCT) values are shown, calculated using the formula: CT(gene of interest) – CT(housekeeping gene Actb). The more positive the dCT value (which is presented on the Y-axis below the zero X-axis), the lower the expression level of the gene of interest. The lower (or more negative) the dCT value, the higher the expression level14,18,21,22. Please click here to view a larger version of this figure.
Pituitary organoid medium (PitOM) | |
Component | Concentration |
Advanced DMEM/F12 | |
Hepes | 1% |
Penicillin-Streptomycin | 1% |
Glutamax | 1% |
B-27 Supplement (50X), minus vitamin A | 1X |
L-Glutamine (200 mM) | 2 mM |
Recombinant Human FGF basic/FGF2/bFGF (157 aa) Protein | 20 ng/mL |
Recombinant Human IGF-1 | 100 ng/mL |
N-2 Supplement (100X) | 1X |
N-acetyl-cysteine | 1.25 mM |
Recombinant Human/Murine FGF-8b | 200 ng/mL |
Recombinant Human FGF-10 | 100 ng/mL |
A83-01 (activin receptor-like kinase 4/5/7 inhibitor) | 0.50 µM |
Recombinant Mouse Sonic Hedgehog/Shh (C25II) N-Terminus | 100 ng/mL |
Recombinant Human EGF Protein, CF | 50 ng/mL |
SB202190 (p38 mitogen-activated protein kinase inhibitor) | 10 µM |
Recombinant Human Noggin | 100 ng/mL |
Cholera Toxin from Vibrio cholerae | 100 ng/mL |
Recombinant Human R-Spondin-1 | 200 ng/mL |
Recombinant Human IL-6 | 20 ng/mL |
Table 1. Composition of PitOM. PitOM is filtered through a 0.22 µm mesh filter and stored at 4 °C for a maximum of 2 weeks.
Medium A | |
Component | Quantity |
DMEM, powder, high glucose | 13.38 g |
HEPES | 5.96 g |
Sodium-Pyruvate (C3H3NaO3) | 0.11 g |
Penicillin G sodium salt | 35.00 mg |
Streptomycin sulfate salt | 50.00 mg |
Sodium Chloride (NaCl) | 0.50 g |
Sodium Hydrogen Carbonate (NaHCO3) | 1.00 g |
Albumin Bovine (cell culture grade) | 3.00 g |
Sterile water | 1.00 L |
Medium C | |
Component | Quantity |
Sodium Chloride (NaCl) | 7.50 g |
Potassium Chloride (KCl) | 0.40 g |
Sodium di-Hydrogen Phosphate 1-hydrate | 0.14 g |
D-glucose | 1.00 g |
HEPES | 4.76 g |
Streptomycin sulfate salt | 50.00 mg |
Penicillin G sodium salt | 35.00 mg |
Phenol red | 10.00 mg |
Albumin Bovine (cell culture grade) | 3.00 g |
Sodium Hydrogen Carbonate (NaHCO3) | 1.00 g |
Sterile water | 1.00 L |
Medium B | |
Component | Quantity |
Titriplex III (Edetate disodium salt dihydrate) | 0.74 g |
Medium C | 100 mL |
Table 2. Composition of medium A, B, and C. All media are filtered through a 0.22 µm mesh filter and stored at 4 °C for a maximum of 4 months. The pH of medium A and C must be adjusted to 7.3.
Cryopreservation medium | |
Component | Concentration |
Advanced DMEM/F12 | 60% |
FBS | 30% |
DMSO | 10% |
Table 3. Composition of cryopreservation medium.
The AL-derived organoids, as described here, represent a powerful research model to study pituitary stem cells in vitro. At present, this organoid approach is the only available tool to reliably and robustly grow and expand primary pituitary stem cells. A pituitary organoid model derived from embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) has been reported previously, which closely recapitulates pituitary embryonic organogenesis23; however, although highly useful to study pituitary development or model pituitary disease23,24,25, the reported protocol, starting from ESC/iPSC, is very time-consuming compared to the protocol described here, and the resulting organoids are also not expandable.
Successful culturing of pituitary stem cell organoids depends on some critical steps in the protocol. It is important to plate an appropriate number of cells at initial cell seeding. A very high number will give rise to overcrowded cultures, which deteriorates the viability of the organoids and obstructs full organoid expansion, whereas a very low number of cells will result in limited organoid formation. Furthermore, it is important not to disturb the integrity of the ECM dome once in culture. Adding and removing medium should be done very carefully, without touching the gel droplet. In addition, prewarming the culture medium reduces the risk of depolymerization of the gel. Finally, passaging the organoids correctly (i.e., dissociating to fragments and not to single cells) is crucial for efficient expansion of the cultures.
These pituitary stem cell organoids can be harnessed to answer questions regarding the stem cells' phenotype, biology, and function. They have already been proven valuable in uncovering novel stem cell features as well as markers of pituitary damage-associated stem cell activation and as a read-out tool for stem cell activity (Figure 1)14,18. Current efforts include their derivation from diseased pituitary, such as hypopituitarism and PitNETs (Figure 1). Eventually, organoids can also be engaged into a platform for drug screening, as successfully established for other diseases26,27. Therefore, further upscaling of the organoid cultures to reach high throughput analysis will be necessary. It has been noticed already that AL-derived organoids can be efficiently grown in a 96-well format, also resulting in more homogenous cultures.
It has been observed that after ~10 passages (corresponding to 3 months of culture), organoid growth efficiency gradually decreased with organoids regrowing at lower numbers and smaller size. This growth decline may be inherent to the intrinsic nature of pituitary stem cells, which may not need to self-renew many times in the gland in vivo, which is only slowly turning over, thus becoming exhausted after a couple of division rounds16,28. Although this eventual growth decline might be considered as a limitation, the model is highly useful since organoid expansion during the preceding passages is more than sufficient for extensive downstream analyses.
Another aspect that might be regarded as a limitation is that the pituitary stem cell organoids do not show prominent differentiation capacity toward the endocrine cell types of the AL, even after xenografting under the kidney capsule of immunodeficient mice (which resulted in a limited number of GH+ and PRL+ cells as described in detail in reference18). Either the right in vitro conditions to drive the stem cells into differentiation are not identified yet, or the major role of the stem cells (especially in the adult gland) is not situated in generating new endocrine cells (since likely not needed in the lazy gland but only in perturbed or challenged conditions)9,10,14,18. Instead, the major function may be situated in other biological aspects (e.g., paracrine signaling to the hormonal progenitor/precursor or mature cells in basic, but likely more in active (developmental, repair, disease) conditions)13,16. Indeed, although pituitary stem cells have been shown to possess multipotent differentiation capacity especially in the embryonic and neonatal period, it is conceivable that stem cells in the adult gland do not (need to) maintain this capacity, given the very low turnover of the adult gland16,28. It is possible that the adult pituitary stem cells act more as a paracrine signaling hub, involved in stimulating or regulating the surrounding progenitor/precursor/endocrine cells13,16. Hence, robust differentiation of the pituitary stem cell organoids culminating in hormone secretion may be an erroneous expectation that will never be reached.
Taken together, the protocol presented here offers a swiftly applicable and reliable tool to robustly expand primary pituitary stem cells in a 3D setting in vitro. The protocol gives rise to organoids that faithfully capture the pituitary stem cell phenotype. The system has already been successfully applied to study pituitary stem cell biology and activation14,18, and the findings are highly translatable to the in vivo situation.
The authors have nothing to disclose.
This work was supported by grants from the KU Leuven Research Fund and the Fund for Scientific Research (FWO) – Flanders. E.L. (11A3320N), and C.N. (1S14218N) are supported by a Ph.D. Fellowship from the FWO/FWO-SB.
2-Mercaptoethanol | Sigma-Aldrich | M6250 | |
48-well plates, TC treated, individually wrapped | Costar | 734-1607 | |
A83-01 | Sigma-Aldrich | SML0788 | |
Advanced DMEM | Gibco | 12491023 | |
Albumin Bovine (cell culture grade) | Serva | 47330 | |
B-27 Supplement (50X), minus vitamin A | Gibco | 12587010 | |
Base moulds | VWR | 720-1918 | |
Buffer RLT | Qiagen | 79216 | |
Cassettes, Q Path Microtwin | VWR | 720-2191 | |
Cell strainer, 40 µm mesh, disposable | Falcon | 352340 | |
Cholera Toxin from Vibrio cholerae | Sigma-Aldrich | C8052 | |
Deoxyribonuclease I from bovine pancreas | Sigma-Aldrich | D5025 | |
D-glucose | Merck | 108342 | |
Dimethylsulfoxide (DMSO) | Sigma-Aldrich | D2650 | |
DMEM, powder, high glucose | Gibco | 52100039 | |
Eppendorf Safe-Lock Tubes, 1.5 mL | Eppendorf | 30120086 | |
Epredia SuperFrost Plus Adhesion slides | Thermo Fisher Scientific | J1800AMNZ | |
Epredia HistoStar Embedding Workstation, 220 to 240Vac | Thermo Fisher Scientific | 12587976 | |
Ethanol Absolute 99.8+% | Thermo Fisher Scientific | 10342652 | |
Fetal bovine serum (FBS) | Sigma-Aldrich | F7524 | |
GlutaMAX Supplement | Gibco | 35050061 | |
HEPES | Sigma-Aldrich | H4034 | |
HEPES Buffer Solution | Gibco | 15630056 | |
InSolution Y-27632 | Sigma-Aldrich | 688001 | |
L-Glutamine (200 mM) | Gibco | 25030081 | |
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-Free | Corning | 15505739 | |
Mr. Frosty Freezing Container | Thermo Fisher Scientific | 5100-0001 | |
N-2 Supplement (100X) | Thermo Fisher Scientific | 17502048 | |
N-Acetyl-L-cysteine | Sigma-Aldrich | A7250 | |
Nunc Biobanking and Cell Culture Cryogenic Tubes | Thermo Fisher Scientific | 375353 | |
Paraformaldehyde for synthesis (PFA) | Merck | 818715 | |
PBS, pH 7.4 | Gibco | 10010023 | |
Penicillin G sodium salt | Sigma-Aldrich | P3032 | |
Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140122 | |
Phenol red | Merck | 107241 | |
Potassium Chloride (KCl) | Merck | 104936 | |
Recombinant Human EGF Protein, CF | R&D systems | 236-EG | |
Recombinant Human FGF basic/FGF2/bFGF (157 aa) Protein | R&D systems | 234-FSE | |
Recombinant Human FGF-10 | Peprotech | 100-26 | |
Recombinant Human IGF-1 | Peprotech | 100-11 | |
Recombinant Human IL-6 | Peprotech | 200-06 | |
Recombinant Human Noggin | Peprotech | 120-10C | |
Recombinant Human R-Spondin-1 | Peprotech | 120-38 | |
Recombinant Human/Murine FGF-8b | Peprotech | 100-25 | |
Recombinant Mouse Sonic Hedgehog/Shh (C25II) N-Terminus | R&D systems | 464-SH | |
RNeasy micro kit | Qiagen | 74004 | |
SB202190 | Sigma-Aldrich | S7067 | |
SeaKem LE Agarose | Lonza | 50004 | |
Sodium Chloride (NaCl) | BDH | 102415K | |
Sodium di-Hydrogen Phosphate 1-hydrate | PanReac-AppliChem | A1047 | |
Sodium Hydrogen Carbonate (NaHCO3) | Merck | 106329 | |
Sodium-Pyruvate (C3H3NaO3) | Sigma-Aldrich | P5280 | |
Stericup-GP, 0.22 µm | Millipore | SCGPU02RE | |
Steriflip-GP Sterile Centrifuge Tube Top Filter Unit, 0.22 μm | Millipore | SCGP00525 | |
Sterile water | Fresenius | B230531 | |
Streptomycin sulfate salt | Sigma-Aldrich | S6501 | |
Syringe, with BD Microlance needle with intradermal bevel, 26G | BD Plastipak | BDAM303176 | |
Thermo Scientific Excelsior ES Tissue Processor | Thermo Scientific | 12505356 | |
Titriplex III | Merck | 108418 | |
TrypL Express Enzyme (1X), phenol red | Thermo Fisher Scientific | 12605028 | |
Trypsin inhibitor from Glycine max (soybean) | Sigma-Aldrich | T9003 | |
Trypsin solution 2.5 % | Thermo Fisher Scientific | 15090046 |