Coexistence of distinct cell types within a shared environment is a frequent phenomenon (Kolenbrander et al., 2002; Pereira-Veiga et al., 2022). For instance, in human blood, various cell types such as bacteria (Kern and Rieg, 2020) and circulating tumor cells (CTCs) (Williams, 2013) may be found, while blood primarily comprises red blood cells (RBCs), white blood cells (WBCs), and platelets (Bain, 2006). Even unicellular species, such as Saccharomyces cerevisiae (S. cerevisiae), can exhibit different phenotypes, with size variations corresponding to different cell cycle phases (Liu et al., 2021b). The separation of specific cell categories with desired characteristics from mixed samples is crucial for various applications (Bankó et al., 2019; Shah and Dickson, 1974). Platelet-enriched plasma, isolated from blood, is known for its efficacy in promoting wound healing (Levoux et al., 2021; Sánchez et al., 2009). Separating bacteria or CTCs from blood can have potential clinical benefits, such as treating septicemia (Lee et al., 2014) or aiding in the diagnosis of cancer metastasis (Nagrath et al., 2007). Furthermore, isolating specific bacteria from polymicrobial communities can enhance research on bacterial identification in complex samples (Pahlow et al., 2015), as well as the understanding of interspecies communication (Federle and Bassler, 2023) and interactions (Fraune et al., 2015). Therefore, cell separation plays a pivotal role in preparing biological and medical assays (Bhagat et al., 2010).

Conventionally, methods such as membrane filtration, centrifugation, and fluorescence-activated cell sorting (FACS) have been employed for cell separation (Cossarizza et al., 2019; Kumar and Lykke, 1984; Sharpe, 1988; Strathmann, 1981; Tanke and van der Keur, 1993). Filter membranes with specific pore sizes allow smaller cells to pass while blocking larger ones, as demonstrated in the separation of Escherichia coli (E. coli) clusters (Zhang et al., 2022). Centrifugation capitalizes on size or density differences between cells, such as the separation of platelets from blood by size using commercial centrifuges (Alves and Grimalt, 2017). FACS combines flow cytometry with cell sorting based on fluorescent labeling, enabling the isolation of various cell types (Pereira et al., 2018). Notably, it has been employed for isolating skeletal muscle stem cells, endothelial cells, hematopoietic cells, and mesenchymal stem cells from muscle tissue (Pasut et al., 2012). Although practical in real-world scenarios, these techniques face limitations such as membrane filtration clogging (Saxena et al., 2009), limited density differences for most cell types in centrifugation (Bracht et al., 2023), and the need for skilled operators in FACS (Bonner et al., 2003).

In the past two decades, microfluidics has witnessed substantial advancements, owing to reduced device scale, improved computer and fabrication technologies, enhanced understanding of particle displacement mechanisms, and interdisciplinary collaboration (Battat et al., 2022; Buriak, 2004; Marre and Jensen, 2010; Riordon et al., 2019; Yager et al., 2006). Microfluidic cell separation techniques fall into two categories: active methods relying on external forces such as acoustic, thermal, electric, and optical forces, and passive methods that primarily modify channel architecture or fluid rheology for particle manipulation (Amirifar et al., 2022; Reece et al., 2016; Zhang et al., 2020). Passive microfluidic cell separation, such as inertial and viscoelastic microfluidics, stands out due to its simplicity and has garnered global attention (Catarino et al., 2019; Liang et al., 2020; Zhang et al., 2021b). For example, Syed et al. demonstrated size-dependent inertial separation of lipid-rich microalgae Tetraselmis suecica (T. suecica) from the invasive diatom Phaeodactylum tricornutum (P. tricornutum) in a spiral microchannel with a trapezoidal cross-section at 1 mL/min (Syed et al., 2018). Liu et al. successfully separated human breast cancer cells MCF-7 from RBCs based on size using polyethylene oxide (PEO) fluid in straight rectangular microchannels at a flow rate of 50 μL/min (Liu et al., 2015). Passive microfluidic cell separation is now extensively studied and has made significant progress (Bayareh, 2020; Shen et al., 2019; Tang et al., 2022).

In this review, our emphasis is directed towards passive microfluidic cell separation techniques, encompassing filtration, sedimentation, pinched flow fractionation (PFF), deterministic lateral displacement (DLD), inertial microfluidics, hydrophoresis, and viscoelastic microfluidics. Besides, we offer insights into hybrid methods that synergistically integrate two or more of these techniques. Typical parameters such as working forces, cell types, separation markers, advantages, and disadvantages of each approach are summarized. Discussion is carried out based on the representative parameters of each separation technique to give a side-by-side comparison between all the approaches. Besides, we provide some general information about the materials and fabrication methods used to produce the microfluidic separation devices in the discussion section. Further, we expound upon the commercial availability of the passive techniques. Future prospects encompassing the development of separation-application or intelligent microfluidic platforms are provided, followed with an overall conclusion for the whole work on further investigations that may be fruitful.