The primary method for organoid culture is 3D cell culture, where stem cells are cultured in a matrix gel with various inhibitors/activators and cytokines. Research is currently focusing on advancing milli-fluidic chip culture and suspension culture, which are dependent on microfluidic devices.

The upcoming sections of this article will delineate the frequently used materials and methodologies in organoid culture. This includes an overview of the typical cell types employed in organoid cultures (Fig. 1) and the development of cell culture systems. And a correct and detailed understanding of the culture methods for normal renal organoids is an important prerequisite for RCC organoid culture methods discussed in this article. Making appropriate modifications in the culture of normal organoids can accurately replicate the tumor microenvironment and preserve the genetic and phenotypic characteristics of RCC.

Fig. 1

This figure summarizes the typical cell types used in organoid cultures, including hESCs, hPSCs, iPSCs, and ASCs

Cell sourcesHuman embryonic stem cells (hESCs)

hESCs, undifferentiated cells derived from the inner cell mass or primordial gonads of early embryos, have the capacity to differentiate into all three primary germ layers of the embryo, leading to the formation of various cell types under specific conditions.

There are many methods to culture organoids from hESCs, including two distinct protocols that have been established for the differentiation of primitive somite (PS) and intermediate mesoderm (IM) into metanephric mesenchyme (MM) and ureteric bud (UB) [9].

The differentiation process involved culturing cells in a medium infused with Vitronectin or Matrigel, which simulates the properties of natural connective tissue. In the first protocol, the medium was supplemented with activin A, bone morphogenetic protein (BMP) 4/7, fibroblast growth factor (FGF) 9, and retinoic acid (RA). The second approach involved the addition of the Wnt agonist CHIR99021 along with FGF9.

Human pluripotent stem cells (hPSCs)

hPSCs possess remarkable self-renewal capabilities and the potential to differentiate into a wide range of somatic cell types and tissues, making them highly valuable for disease modeling and regenerative applications. A highly efficient and serum-free process for differentiating hPSCs into ureteric bud (UB) organoids and functional collecting duct (CD) cells involves several steps with L-GlutaMAX, FGF9 and CHIR99021 (Fig. 2). Initially, hPSCs are induced to form pronephric progenitor cells (NPCs),, which then aggregate into spheres resembling the nephric duct. In a 3D matrix, these spheres evolve into UB organoids, displaying branching morphogenesis [10]. In another approach, hPSCs are cultured in a ReproFF2 medium forming well-defined circular colonies. These undifferentiated hPSCs are isolated and passaged every seven days. For inducing NPCs, Advanced RPMI 1640 medium with L-GlutaMAX and FGF9 is employed, followed by the addition of CHIR99021 to generate structures akin to renal vesicles [11]. A successful method selectively induces metanephric NPCs, which can develop into in vivo counterparts. The combination of activin, GSK-3β inhibitor CHIR99021, and FGF9 further directs NPCs to form renal vesicles and late primitive streak, eventually giving rise to posterior intermediate mesoderm. These structures can self-organize into nephronic structures, expressing markers of podocytes, proximal tubules, Henle’s loops, and distal tubules, arranged in a continuous, organized manner [12].

Fig. 2

This figure summarizes the protocol using L-GlutaMAX, FGF9 and CHIR99021 that culture organoids from hPSCs and temporal stages of the organoid development of renal organoids

The efficiency of these differentiation methods is influenced by the inherent variability of hPSC lines because the cells in the interstitial space of kidney organoids are not yet fully characterized.

Induced pluripotent stem cells (iPSCs)

iPSCs, the primary source for organoid formation, are derived by reprogramming differentiated cells from patients or healthy individuals. Organoids created from iPSCs can model hereditary or infectious kidney diseases, making iPSCs a key resource for organoid culture.

Taguchi and Nishinakamura [13] determined that a combination of retinoic acid (RA), CHIR99021 at a low concentration (1 µM), and FGF9 (5 ng/mL), either with or without glial cell line-derived neurotrophic factor (GDNF), an attractant similar to Wnt11, is optimal for inducing differentiation. This mix promotes the anterior intermediate mesoderm (AIM) from mouse or human iPSCs to undergo a mesenchymal-to-epithelial transition (MET), leading to the formation of the epithelialized Wolffian duct (WD). Ultimately, this process results in the development of the UB with the ability to branch.

Musah’s team developed a method to differentiate human iPSCs into mesoderm cells using activin A, CHIR99021, and Rho kinase (ROCK) inhibitor Y27632. Subsequently, BMP7 and CHIR99021 were used to induce the formation of intermediate mesoderm cells. These cells were then cultured in a medium containing BMP7, activin A, vascular endothelial growth factor (VEGF), retinoic acid (RA), and CHIR99021 for 4 to 5 days to successfully generate podocytes [14].

Controllable 3D growth environments are crucial for enhancing the consistency and maturity of organoids. Consequently, iPSC-derived kidney organoids were matured in fully synthetic self-assembling peptide hydrogels (SAPHs) with varying degrees of stiffness. This minimally complex microenvironment is tailored for kidney differentiation. As a result, organoids with high viability were produced, although a slight decrease in cell viability was observed with increased matrix stiffness [15].

Adult stem cells (ASCs)

ASCs, found in differentiated tissues, have the ability to self-renew and differentiate. Fresh tissue from patients with clear cell RCC was used to develop air-liquid interface (ALI) patient-derived organoids (PDOs). This method resulted in organoids that resembled the tumor histology and immune microenvironment [16, 17]. Organoid growth was initially achieved with fetal calf serum and enhanced by adding growth factors like WNT3A, EGF, NOGGIN, and RSPO1 (WENR). Passaging of ALI PDOs was performed by addition of 200 units collagenase IV for 30 min at 37 °C until the collagen was dissociated. Then, three washes with PBS and EDTA were performed to inhibit collagenase activity. The ALI PDOs were absorbed with 1 ml of type I collagen solution and replicated into a new ALI collagen gel at the desired mass density. These ALI PDOs, with a solid growth pattern, remained viable for over 30 days in culture.

Another approach mixed tumor cells with a cold basement membrane extract containing EGF. In the study of Li et al. [18], organoids were passaged every 2–3 weeks at a split ratio of 1:2–1:3, leading to the generation of renal cancer organoid lines such as ccRCC, pRCC, and chRCC.

In another protocol, cells were also maintained in a serum-free medium with EGF and basic fibroblast growth factor (bFGF). They were then cultured in a medium with low-density Matrigel, EGF, bFGF, ROCK inhibitor, and A8301, an inhibitor of TGF-β, ALK4, and ALK7 [19].

Organoids from ASCs have a short cultivation cycle and high genetic stability but lack interstitial and endothelial cells, marking them as primarily epithelial cell systems.

Urine-derived stem cells (USCs)

Compared to adult somatic stem cells and human pluripotent stem cells, urine-derived stem cells (USCs) can be obtained through a non-invasive method, and have been proven to possess regenerative properties. They exhibit robust proliferative potential and can successfully differentiate into urothelial lineages. Organoids differentiated from USCs have been applied in research on hereditary kidney diseases and motor neuron diseases [20].

The study by Liang Chen et al. [21] indicates that USC-organoids share similar morphological, histological, gene expression profiles, and nephrotoxicity screening capabilities with kidney organoids derived from other sources. Research by Wan et al. [20] also notes that urothelial organoids produced from stem cells isolated from urine resemble natural urothelial organoids in phenotype and function. In summary, USC-organoids not only face fewer ethical restrictions but also possess comprehensive scientific value. Although the collection process may be more time-consuming compared to adult somatic stem cells and human pluripotent stem cells, the non-invasive advantage makes them a promising adjunct for personalized treatment of renal cancer patients in future clinical applications.

Establishment of cell culture system

Traditional media used for cultivating cancer organoids generally include components such as B-27 supplement, nicotinamide, R-spondin1, noggin, N-acetyl-l-cysteine, A83-01, SB202190, FGF 10, EGF, and Y-27,632 [22, 23]. Building on this foundation, a variety of innovative cultivation systems have been developed, encompassing adherent 3D cell culture, milli-fluidic chip culture, and suspension culture reliant on microfluidic devices (Fig. 3).

Fig. 3

This figure focuses on a variety of organoid culture systems, including adherent 3D cell cultures, milli-microfluidic chip cultures, and suspension cultures dependent on microfluidic devices

Adherent 3D cell culture

3D multicellular miniature organoids are extensively used for mimicking organ development and disease progression. The extracellular matrix (ECM) plays a vital role in the self-renewal and differentiation of stem cells by providing a necessary scaffold for cell adhesion and growth during organoid culture. Hydrogels, particularly Matrigel, are frequently utilized as scaffolds to support cell proliferation in organoid cultures, facilitating the formation of complex structures that resemble corresponding organs and preserve their physiological structure and functional traits.

In Low et al. [24], cells were dissociated using Accutase and then aggregated in a basal differentiation medium. This process initially generated epiblast spheroids, which subsequently developed into kidney tubules. After cell dissociation, the cells, when sandwiched between two layers of diluted Matrigel, formed compact, ball-like colonies and then developed internal cavities [25].

Milli-fluidic chip culture

Renal organoids generated from hPSCs feature sections resembling glomeruli and tubules, but these components often remain underdeveloped in static cultures. The application of microfluidics technology significantly addresses this challenge. By cultivating kidney organoids in an environment with fluid flow and vascularization, the development of nephron structures are notably enhanced.

An in vitro technique for culturing kidney organoids on a millifluidic chip significantly enhances the expansion of their intrinsic endothelial progenitor cells and fosters the development of vascular networks with perfusable lumens encircled by mural cells. The millifluidic chip is characterized by its high flux and small volume, enabling sample and reagent conservation. It also facilitates real-time monitoring and manipulation. The application of superhydrophobic coatings on the chip’s surface creates distinct physical separations between each micropore, ensuring each one has its own isolated liquid environment. This design simplifies the addition and alteration of reagents.

In comparison to static cultures, vascularized renal organoids cultured under flow conditions exhibited more advanced development in their podocyte and tubular compartments, which is required to facilitate functional morphogenesis of podocytes in vitro. This advancement was characterized by enhanced cellular polarity and a more mature adult gene expression profile [26].

Suspension culture dependent on microfluidic device

Microfluidic techniques, known for their high precision, reduced contamination risk, and user-friendly operation, commonly utilize cell-laden alginate hydrogel beads. These beads are biologically inert and are used in suspension cultures through various methods such as microplate, pendant drop, rotational culture, and magnetic suspension.

Human renal cancer cells were incorporated into alginate hydrogels using a straightforward and cost-effective microfluidic device which was designed in a computer-aided design software as a three-layer structure. The top, middle, and bottom layers show the inlet and outlet holes, the outline of the fluidic channel, and the lower boundary of the microfluidic device in two dimensions, respectively. This device facilitates the infusion of various fluids, including cell solutions, alginate, calcium chloride, and mineral oil, to produce water-in-oil droplets for size-controllable organoid culture [27]. This technique enables the rapid and economical creation of uniformly sized organoids, suitable for replicable experimental setups.