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1, BME-2, or collagen) for subsequent culture, enables a co-culture system of mesenchymal stem cells (MSC) and
continuous optimization of growth conditions. 31 peripheral blood mononuclear cells (PBMC), forming the
MSC-PDO-PBMC model. Preliminary applications in
2.2.2.2. Microfluidic cultivation drug experiments, including chemotherapy and molecular
Microfluidics, a technology for manipulating and processing targeted therapies, have demonstrated the potential of this
liquid flow on a micrometer scale, has demonstrated model in drug screening and cancer research. 35
significant potential in cultivating organoids in recent years. (v) Microenvironmental regulation
This technology is particularly effective for simulating the Microfluidics influences the development and
microenvironment of organs and controlling conditions for cell functionality of organoids by regulating microenvironmental
growth and differentiation. The following are several primary factors such as temperature, pH, and oxygen concentration,
modes of organoid cultivation using microfluidic technology: thereby providing culture conditions that more closely
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(i) Monolayer cell culture resemble physiological states. This technology opens
By employing a microfluidic chip design, cells can form new possibilities in developing functional retinal tissue
a monolayer membrane structure within microchannels. engineering by guiding the connectivity of various cell
Through precise control of liquid flow, the required oxygen populations. The design of microchannels can direct
and nutrients for cell growth can be maintained, while axonal connections to grow exclusively in a single
metabolic waste is effectively eliminated. Microfluidic- direction, mimicking the conditions found in native tissues.
mediated 2D monolayer gastric organoids can rapidly Furthermore, the integration of valves and pumping
spread to form a uniform cell layer, offering an advantage systems facilitates the functional maturation and long-term
for simulating the upward-growing columnar epithelium viability of the tissues, addressing one of the main challenges
found in the gastric wall. 32 currently faced by 3D tissue engineering technologies. 37
(ii) Three-dimensional culture 2.2.2.3. Air-liquid interface (ALI) culture
By utilizing 3D structures within microfluidic chips,
cells naturally form aggregates in the microenvironment, The ALI method is a technique that involves cultivating cells
mimicking the 3D structure of organs. Microfluidic devices at the boundary between gas and liquid phases, which has
can provide uniform liquid flow and nutrient distribution, garnered significant attention and widespread application
supporting long-term cell growth and differentiation. in organoid research in recent years. In contrast to
This method is highly suitable for cultivating complex 3D conventional submerged culture methods, this cultivation
organoids, such as liver organoids. 33 paradigm provides a more precise representation of in vivo
conditions. It is significant for constructing epithelial cell
(iii) Dynamic fluidic culture organoids with specific functional and structural attributes.
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Dynamic fluid flow can be achieved through The ALI culture model cultivates cells using a porous
microfluidic systems, enabling continuous or pulsatile fluid support system, where the bottom of the cells is immersed
movement that mimics physiological conditions, such as in a culture medium while the top remains exposed to air.
blood or lymphatic flow. This dynamic environment aids This air-liquid interphase environment encourages cells to
in maintaining the physiological state of cells and promotes grow upward, forming a multilayered cellular architecture
the maturation of organoids. For instance, microfluidic conducive to the differentiation and maturation of neurons.
devices can construct a sustained tumor microenvironment, Brain organoids cultured at the air-liquid interphase
facilitating interactions between tumor fragments exhibit enhanced electrophysiological activity, including
and allowing a continuous flow of tumor-infiltrating inter-neuronal connectivity and signal transmission,
lymphocytes. This setup simulates lymphocyte-mediated which is important for studying brain function and disease
tumor immunity and infiltration, providing valuable models. Furthermore, this model is well-suited for
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insights into cancer research and immunotherapy. 34 cultivating epithelial organoids, such as gastrointestinal or
(iv) Multi-layered fluidic culture respiratory organoids, as these tissues naturally develop in
The architecture of multi-layer fluidic channels within an environment characterized by the interaction between
a microfluidic chip allows different types of cells or culture gas and liquid in vivo. 40,41 Biocompatible porous materials,
media to flow in distinct layers, thereby enabling cellular such as collagen, sodium alginate, or polylactic acid, are
isolation or interaction. By regulating the fluid flow in these often used as scaffolds during cultivation. These materials
layers, it is possible to simulate the multi-layered structures support cell attachment and facilitate the exchange of
found in organs. For instance, in the construction of patient- nutrients and gases. In summary, the ALI culture method,
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derived organoid (PDO) models for hepatocellular carcinoma, with its efficient oxygen supply and spatial utilization,
the tumor microenvironment necessary for organoid growth has emerged as a crucial cultivation technique in modern
is created by the multi-layer microfluidic chip, which houses bioscience and engineering research.

Volume 1 Issue 2 (2025) 4 doi: 10.36922/or.8262