A. Top-down technologies for controlled tissue patterning

1. 3D photopatterning

Light is an attractive stimulus for the patterning of cells and tissues as it is noninvasive, and the intensity and spatial distribution of the stimulus can readily be modulated to control patterning. Specifically, photopatterning can be used to control hydrogel cross-linking and degradation or the presentation and release of biochemical cues, such as adhesive motifs and growth factors. Combining moieties that are responsive to different wavelengths of light can also provide fine-tuning of multiple parameters within a single system, allowing the generation of quite complex patterns. Irrespective of whether photopatterning seeks to modulate the biomaterial cross-linking, degradation, or presentation of biochemical cues, simple photopatterning can be achieved via the use of a photomask to selectively illuminate regions of a sample [Fig. 2(a)]. While this is more suited to patterning across a 2D surface, it has been also used to create simple 3D channels through the depth of a hydrogel.2727. C. A. DeForest, B. D. Polizzotti, and K. S. Anseth, Nat. Mater. 8, 659 (2009). https://doi.org/10.1038/nmat2473 More complex 3D patterning can be achieved by two-photon lithography—by focussing the light into a point, this approach allows patterns to be “written” into the 3D hydrogel [Fig. 2(b)]. An advantage of two-photon patterning is that excitation depends on the local light power density, meaning that the volume in which photopatterning actually happens is small and can generate very high-resolution features. Overall, the field of photopatterning is relatively mature, with many strategies and chemistries available.28,2928. T. E. Brown and K. S. Anseth, Chem. Soc. Rev. 46, 6532 (2017). https://doi.org/10.1039/C7CS00445A29. L. Li, J. M. Scheiger, and P. A. Levkin, Adv. Mater. 31, 1807333 (2019). https://doi.org/10.1002/adma.201807333 More recent attention is on the capacity to control multiple aspects simultaneously (i.e., structure and functionalization) or for dynamic control, providing opportunities for spatiotemporal or 4D patterning.2929. L. Li, J. M. Scheiger, and P. A. Levkin, Adv. Mater. 31, 1807333 (2019). https://doi.org/10.1002/adma.201807333One of the critical aspects to achieve highly controlled photopatterning is the selection of the hydrogel material. For instance, hydrogels based on gelatin methacryloyl (GelMA) are very widely used for 3D cell culture by means of light-induced cross-linking. Functionalization of the gelatin polymer with methacrylate groups facilitates cross-linking via free-radical-initiated chain polymerization, when in the presence of a photoinitiator and illuminated under the relevant wavelength of light.3030. C. Yu et al., Chem. Rev. 120, 10695 (2020). https://doi.org/10.1021/acs.chemrev.9b00810 Variations of this chemistry, such as hyaluronic acid (HA)-methacrylate or poly-ethylene glycol (PEG)-diacrylate (PEGDA), have also been widely adopted.30–3230. C. Yu et al., Chem. Rev. 120, 10695 (2020). https://doi.org/10.1021/acs.chemrev.9b0081031. H. Mutlu et al., Macromol. Rapid Commun. 40, 1800650 (2019). https://doi.org/10.1002/marc.20180065032. J. R. Choi, K. W. Yong, J. Y. Choi, and A. C. Cowie, Biotechniques 66, 40 (2019). https://doi.org/10.2144/btn-2018-0083 Bio-orthogonal click reactions such as the thiolene photo-click reaction also present an attractive strategy due to their fast reaction conditions and ability to be performed under mild “cell-friendly” conditions, for example, by creating thioether bonds between PEG-dithiol and norbornene-functionalized gelatine.33,3433. J. W. Nichol, S. T. Koshy, H. Bae, C. M. Hwang, S. Yamanlar, and A. Khademhosseini, Biomaterials 31, 5536 (2010). https://doi.org/10.1016/j.biomaterials.2010.03.06434. T. P. T. Nguyen et al., Biomaterials 279, 121214 (2021). https://doi.org/10.1016/j.biomaterials.2021.121214 Conversely, photodegradable hydrogels can be produced by incorporating photolabile groups into hydrogel during cross-linking, which then undergo scission when exposed to light. Common photodegradable moieties are ortho-nitrobenzyl (ONB) and coumarin, which have been widely used to both degrade and soften hydrogel networks, but can also be used to expose functional groups for site-specific conjugation of biomolecules.3535. V. X. Truong, ChemPhotoChem 4, 564 (2020). https://doi.org/10.1002/cptc.202000072As a relatively mature technology, photopatterning has been widely used to generate structures within hydrogels that guide cell migration and distribution [Figs. 2(c)-2(e)]. This can be achieved by selectively degrading areas of a bulk hydrogel3636. M. W. Tibbitt, A. M. Kloxin, K. U. Dyamenahalli, and K. S. Anseth, Soft Matter 6, 5100 (2010). https://doi.org/10.1039/c0sm00174k or by controlling the cross-linking to create specific structures37,3837. A. Ovsianikov et al., Biomacromolecules 12, 851 (2011). https://doi.org/10.1021/bm101530538. A. Zennifer, S. Manivannan, S. Sethuraman, S. G. Kumbar, and D. Sundaramurthi, Mater. Sci. Eng. C 134 112576 (2021). https://doi.org/10.1016/j.msec.2021.112576 (see on bioprinting). Initially, experiments in 2D showed the power of physical structures to guide cell organization. For example, the alignment of cultured cardiac fibroblasts and cardiomyocytes was improved when grooves were patterned into photodegradable GelMA, with positive effects on contractility and beating synchronicity.3939. G. Gilles, A. D. McCulloch, C. H. Brakebusch, and K. M. Herum, PLoS One 15, e0241390 (2020). https://doi.org/10.1371/journal.pone.0241390 3D photopatterning has been used widely to create 3D channels and pores, aimed toward the creation of vasculature models. One example is the work by Brandenburg et al.,4040. N. Brandenberg and M. P. Lutolf, Adv. Mater. 28, 7450 (2016). https://doi.org/10.1002/adma.201601099 in which quite complex 3D structures, including spirals and irregular shapes, were created via light-mediated degradation. Branched channel structures, replicating complex vascular networks, were produced and seeded with human umbilical vein endothelial cells (HUVECs) to create an artificial vasculature. However, a disadvantage of this work was the high intensities of light required for degradation, which can pose issues with cell viability. Later studies have used wavelengths and intensities of light that can create structures with less possibility of damaging cells and tissues. Arakawa et al.2525. C. K. Arakawa, B. A. Badeau, Y. Zheng, and C. A. DeForest, Adv. Mater. 29, 1703156 (2017). https://doi.org/10.1002/adma.201703156 generated channels via two-photon lithography by combining PEG with an RGD peptide cross-linker that also included an ONB ester. The ONB facilitated photodegradation upon exposure to near infrared (740 nm) light and channels for vascularization were created by two-photon lithography. This technique was successful in generating defined patterns of vasculature (HUVEC-lined channels) within a bulk hydrogel populated by stromal cells. Alternatively, Khetan et al. used a photomask to pattern nondegradable areas into a hydrogel for cell-directed degradation.4141. S. Khetan and J. A. Burdick, Biomaterials 31, 8228 (2010). https://doi.org/10.1016/j.biomaterials.2010.07.035 First, a uniform hydrogel was made using Michael addition to cross-link HA with adhesive RGD-peptide and degradable MMP-cleavable peptides. Nondegradable kinetic chains were selectively included via a UV light-induced secondary radical reaction, producing specific areas that could be degraded by cells or not. These resulted in control of infiltration of chick limb cells (to the degradable areas) and also differences in mesenchymal stromal cell (MSC) differentiation bias.Alternatively, light has been used to locally modify hydrogel elastic modulus, patterning stiff, and soft regions into a substrate. Although studies are, thus far, limited to 2D hydrogels, current data show the power that these changes can have in dictating cell patterning. For example, RAW 264.7 macrophages preferentially clustered in areas of high elastic modulus when cultured on PEGDA hydrogels in which specific areas had been softened by photodegradation.4242. S. Nemir, H. N. Hayenga, and J. L. West, Biotechnol. Bioeng. 105, 636 (2010). https://doi.org/10.1002/bit.22574 Similarly, patterning of methacrylated HA hydrogels into checkerboard (50 × 50 or 100 × 100 μm2) and striped (500 μm) patterns revealed effects on focal adhesion formation and proliferation in bovine aortic endothelial cells, which has relevance to matrix stiffening in artherosclerosis4343. M. C. Lampi, M. Guvendiren, J. A. Burdick, and C. A. Reinhart-King, ACS Biomater. Sci. Eng. 3, 3007 (2017). https://doi.org/10.1021/acsbiomaterials.6b00633 Patterning of elastic modulus can also affect MSC fate, as demonstrated by Yang et al.,4444. C. Yang et al., Proc. Natl. Acad. Sci. U.S.A. 113, E4439 (2016). https://doi.org/10.1073/pnas.1609731113 who used photodegradation to present MSCs with varying percentage area and patterning of stiff (10 kPa) and soft (2.3 kPa) PEGDA substrates. The ratio of soft:stiff adhesive area and the patterning within this dictated the degree of cell spreading, cytoskeletal architecture, and osteogenic differentiation.Patterning of biochemical cues is another key means to control cell fate. This has been widely investigated for coupling of cell-adhesive factors, such as RGD, but can also be used to tether growth factors or cytokines. The simplest approach is to use the same chemistry as for the initial hydrogel cross-linking, as long as some functional groups remain postgelation. In this strategy, pre-crosslinked hydrogels are immersed in a solution of functionalized-peptide that react with remaining functional groups in the hydrogel upon illumination. Examples include pattering of diacrylate-functionalized RGD in PEGDA hydrogels4545. S.-H. Lee, J. J. Moon, and J. L. West, Biomaterials 29, 2962 (2008). https://doi.org/10.1016/j.biomaterials.2008.04.004 and cysteine-terminal peptides in norbornene-functionalized PEG hydrogels.27,4627. C. A. DeForest, B. D. Polizzotti, and K. S. Anseth, Nat. Mater. 8, 659 (2009). https://doi.org/10.1038/nmat247346. B. D. Fairbanks, M. P. Schwartz, A. E. Halevi, C. R. Nuttelman, C. N. Bowman, and K. S. Anseth, Adv. Mater. 21, 5005 (2009). https://doi.org/10.1002/adma.200901808 Uncaging of reactive groups is also a common strategy. Here, similar chemistries are used as in patterning by photodegradation, but the light removes a protective motif, rather than breaking bonds to degrade the hydrogel structure. An early exemplar from the Shoichet group generated agarose hydrogels containing ONB-protected cysteine groups. Uncaging of the cysteine afforded coupling to maleimide-functionalized RGD peptide upon illumination with UV light.4747. Y. Luo and M. S. Shoichet, Nat. Mater. 3, 249 (2004). https://doi.org/10.1038/nmat1092 This uncaging strategy has been widely used across varying chemistries, including ONB, coumarin, and cyclopropenone.27,4827. C. A. DeForest, B. D. Polizzotti, and K. S. Anseth, Nat. Mater. 8, 659 (2009). https://doi.org/10.1038/nmat247348. Z. Cai et al., Chem. Mater. 31, 4710 (2019). https://doi.org/10.1021/acs.chemmater.9b00706 It has also been extended to enzymatic coupling, as exemplified by Mosiewicz et al.,4949. K. A. Mosiewicz et al., Nat. Mater. 12, 1072 (2013). https://doi.org/10.1038/nmat3766 where light was used to uncage the substrate for transglutaminase factor XIII (K-peptide), which was integrated into a PEG hydrogel network. An adaptation of this process can be used to release tethered molecules, such as the removal of RGD.5050. D. Ma, N. Zhou, T. Zhang, K. Hu, X. Ma, and N. Gu, Colloids Surf., A 522, 97 (2017). https://doi.org/10.1016/j.colsurfa.2017.02.073 More complex regimes seek to pattern multiple cues or reversible pattern biomolecules. As an example, the dual-patterning of sonic hedgehog and ciliary neurotrophic factor was achieved by sequentially coupling streptavidin and then barstar tags with uncaged thiol groups in an agarose hydrogel. When incubated in a protein solution, this then enabled coupling of biotin-conjugated ciliary neurotrophic factor and barstar-conjugated sonic hedgehog, respectively.5151. R. G. Wylie, S. Ahsan, Y. Aizawa, K. L. Maxwell, C. M. Morshead, and M. S. Shoichet, Nat. Mater. 10, 799 (2011). https://doi.org/10.1038/nmat3101Dynamic patterning of biochemical cues can be achieved by reversible photocoupling to mirror the dynamic changes that occur throughout development or by selectively releasing signaling proteins that would be unstable in solution over longer time periods.5252. D. R. Griffin, J. L. Schlosser, S. F. Lam, T. H. Nguyen, H. D. Maynard, and A. M. Kasko, Biomacromolecules 14, 1199 (2013). https://doi.org/10.1021/bm400169d DeForest et al.5353. C. A. DeForest and K. S. Anseth, Nat. Chem. 3, 925 (2011). https://doi.org/10.1038/nchem.1174 demonstrated this by reversibly coupling RGD into PEG hydrogels via thiolene photoaddition of a cysteine-containing RGD peptide and photodegradation via ONB. A limitation of this approach was that it was only suitable for conjugation of simple molecules, such as short peptides. However, full proteins have now been reversibly patterned using light to deprotect an oxime-ligation sequence for protein coupling, while scission of an ONB ester releases the protein.5454. I. Batalov, K. R. Stevens, and C. A. DeForest, Proc. Natl. Acad. Sci. U.S.A. 118, e2014194118 (2021). https://doi.org/10.1073/pnas.2014194118 Interestingly, protein removal reinstates the functional groups required for coupling meaning that a second protein can then be patterned into blank areas created.Finally, an alternative approach is to use optogenetics to provide precise spatial and temporal regulation of signaling pathways that are key drivers of tissue patterning. The light stimulus does not alter biomaterial properties or biomolecule availability but instead relies upon optogenetic tools like photoactivatable receptors, kinases, and transcriptional activators to control cell fate and tissue organization in both time and space.5555. D. Krueger, E. Izquierdo, R. Viswanathan, J. Hartmann, C. Pallares Cartes, and S. De Renzis, Development 146, dev175067 (2019). https://doi.org/10.1242/dev.175067 With origins in control of neurons, optogenetics has been used to control neurite outgrowth and cell-cell connectivity5656. S. Park et al., Sci. Rep. 5, 9669 (2015). https://doi.org/10.1038/srep09669 The technique has since found application in the control of many cell types. Precise spatial and temporal control over a photoactivatable Rho kinase in the drosophila embryo triggered myosin-mediated cell contraction and tissue folding5757. E. Izquierdo, T. Quinkler, and S. De Renzis, Nat. Commun. 9, 2366 (2018). https://doi.org/10.1038/s41467-018-04754-z Similarly, modulation of a photoactivatable receptor for signaling protein Nodal demonstrated how changes in the initiation and duration of Nodal signaling can be used to promote and repress formation of cell and tissue types in vertebrate embryos5858. K. Sako et al., Cell Rep. 16, 866 (2016). https://doi.org/10.1016/j.celrep.2016.06.036 A creative example of optogenetics in cell patterning used light to initiate heterodimerization of Cre recombinase. This then activated expression of MyoD promoting myogenic differentiation to skeletal myoblasts, which could be patterned by a photomask.5959. L. R. Polstein, M. Juhas, G. Hanna, N. Bursac, and C. A. Gersbach, ACS Synth. Biol. 6, 2003 (2017). https://doi.org/10.1021/acssynbio.7b00147 Optogenetics has also been used to pattern cardiac tissue. Here, light was used to activate a Wnt receptor and control pluripotent cell differentiation into cardiomyoctes, endothelial progenitors, and epicardial cells.6060. P. B. Hellwarth et al., Adv. Genet. 2, e202100011 (2021). https://doi.org/10.1002/ggn2.202100011 Advantages of optogenetics include the ability to pattern with high spatial resolution (μm) and control of timing to less than 1 ms.6161. O. A. Shemesh et al., Nat. Neurosci. 20, 1796 (2017). https://doi.org/10.1038/s41593-017-0018-8 Thus, optogenetic tools could provide an additional layer of control to guide cell fate and tissue patterning.While technologies to photopattern hydrogels are relatively mature, and the field has developed strategies for exquisite spatiotemporal control of the microenvironment, the application of these to biological systems still has much scope. One current limiting factor is the need to improve the reproducibility and mechanical stability of the hydrogels, particularly if the patterned constructs are to be integrated into culture-on-a-systems, where microfluidics are used to provide physiologically relevant forces such as fluid flow and shear stress. On the biological side, many current exemplars are so far limited to applications in vascularization and cell outgrowth (such as neurite extension), although there are some reports of effects on stem cell differentiation and macrophage polarization47,54,6247. Y. Luo and M. S. Shoichet, Nat. Mater. 3, 249 (2004). https://doi.org/10.1038/nmat109254. I. Batalov, K. R. Stevens, and C. A. DeForest, Proc. Natl. Acad. Sci. U.S.A. 118, e2014194118 (2021). https://doi.org/10.1073/pnas.201419411862. Y. Luo, X. Zheng, P. Yuan, X. Ye, and L. Ma, Bioact. Mater. 6, 4065 (2021). https://doi.org/10.1016/j.bioactmat.2021.04.018 Future efforts are likely to focus on extending these technologies toward the patterning of more complex tissues and organoids. For example, the Lutholf group that has used biomaterials to pattern intestinal organoids using photodegradation of PEG-based hydrogels was to selectively soften specific areas.26,6326. N. Gjorevski et al., Science (80-) 375, eaaw9021 (2022). https://doi.org/10.1126/science.aaw902163. M. Nikolaev et al., Nature 585, 574 (2020). https://doi.org/10.1038/s41586-020-2724-8 This facilitated budding of the organoid and formation of cryptlike structures, allowing sites of budding to be specified. Studies such as this play an important role in identifying the role of tissue architecture and geometry in development. Recent attention toward the development of moieties that can be activated with longer wavelengths of light to decrease the potential for cytotoxic effects and improve the depth of penetration into a 3D sample will also yield new opportunities. Overall, the field of 3D hydrogel photopatterning has developed many valuable tools with which to study and control cell patterning and is set to make an enormous contribution to cell patterning efforts into the future.

2. Magnetism

Magnetic manipulation of cells involves applying an external magnetic field that is used to dictate their position in 3D space. Specifically, a cell's migration in response to a magnetic field is referred to as magnetophoresis. A key benefit of this method is that the external magnetic field can be applied remotely without disturbing the tissue construct or cells directly. As described by Yaman et al.,6464. S. Yaman, M. Anil-Inevi, E. Ozcivici, and H. C. Tekin, Front. Bioeng. Biotechnol. 6, 192 (2018). https://doi.org/10.3389/fbioe.2018.00192 current magnetophoresis technologies can be clustered into negative or positive magnetophoresis methods. When cells respond to the force of a magnetic field and align themselves to the location with the highest field strength, positive magnetophoresis occurs. When cells respond by moving away from locations of high field strengths, negative magnetophoresis occurs. While for positive magnetophoresis, the cells must be susceptible to magnetic actuation; in negative magnetophoresis, only the medium in which the cells are has to be magnetic.A main drawback for positive magnetophoresis is that only few cell types are intrinsically magnetic and the majority of cells need to be magnetically labelled before this technique can be applied6565. B. D. Plouffe, S. K. Murthy, and L. H. Lewis, Rep. Prog. Phys. 78, 016601 (2015). https://doi.org/10.1088/0034-4885/78/1/016601 (Fig. 3). An example of cells that are intrinsically magnetic are human erythrocytes, whose covalently bound iron atom in the hemoglobin subunits confers magnetic properties. When deoxygenated, hemoglobin displays paramagnetic behavior due to the charge on the unbound iron atom. When oxygenated, hemoglobin is diamagnetic. This can be used to manipulate cell positioning, as demonstrated by Zborowski et al.,7070. M. Zborowski, G. R. Ostera, L. R. Moore, S. Milliron, J. J. Chalmers, and A. N. Schechter, Biophys. J. 84, 2638 (2003). https://doi.org/10.1016/S0006-3495(03)75069-3 who exploited this phenomenon to demonstrate significant mobility of deoxygenated erythrocytes in the presence of a magnetic field, compared to oxygenated erythrocytes. Conversely, Fattah et al. utilized oxygenated whole blood to generate spheroids and other patterns in situ7171. A. R. Abdel Fattah et al., ACS Biomater. Sci. Eng. 2, 2133 (2016). https://doi.org/10.1021/acsbiomaterials.6b00614—erythrocytes were suspended in a paramagnetic solution, and when a magnetic field was applied, the cells were circulated by the paramagnetic medium to a location with the lowest field strength.Cells that are not intrinsically magnetic can be labeled for magnetic actuation by loading them with magnetic nanoparticles (MNPs). In one example, this was achieved via super paramagnetic iron oxide nanoparticles (SPIONs), which were endocytosed by endothelial cells Fig. 3(c).6868. B. R. Whatley, X. Li, N. Zhang, and X. Wen, J. Biomed. Mater. Res. Part A 102, 1537 (2014). https://doi.org/10.1002/jbm.a.34797 A prefabricated pattern in a magnetic material was used to arrange the endothelial cells into spheroids, which were then manipulated into larger 3D structures. Importantly, a follow-up to this work6868. B. R. Whatley, X. Li, N. Zhang, and X. Wen, J. Biomed. Mater. Res. Part A 102, 1537 (2014). https://doi.org/10.1002/jbm.a.34797 determined optimal SPION concentrations within the endothelial cells and showed that they did not introduce cytotoxicity or diminish cell proliferation. 3D embryoid bodies (EB) were then created in a controlled manner and allowing the impact of the size and shape of 3D multicellular structures on differentiation profiles to be examined. Du et al.7272. V. Du et al., Nat. Commun. 8, 400 (2017). https://doi.org/10.1038/s41467-017-00543-2 also used endocytosis of MNPs and magnetic forces to aggregate embryonic stem cells into 3D EBs without the need for a scaffold. This permitted a greater control of EB size as compared to conventional methods, and with the addition of a second magnet, the EBs could be deformed with remote magnetic forces to stretch and mechanically stimulate cells at will. Cyclic stretching (mimicking heart muscle contraction) was shown to promote cardiac differentiation without the need for chemical factors, demonstrating its use as a tool to explore the impact of physical biosensing on ESCs differentiation. MNPs can also be encapsulated, which can improve their accumulation within the cell via electrostatic interactions occurring at the cell surface. Ino et al.6767. K. Ino, A. Ito, and H. Honda, Biotechnol. Bioeng. 97, 1309 (2007). https://doi.org/10.1002/bit.21322 used magnetic cationic liposomes (MCLs)—liposomes containing 10 nm magnetite nanoparticles—to label mouse fibroblasts (FBs) and HUVECs. By placing the culture dish onto a steel plate, a magnet could be used to pattern both cell types into patterns as complex as the shape of the letters “M,” “A,” and “G” [Fig. 3(b)]. Curved patterns with FBs and cordlike structures using HUVECs were also demonstrated. Importantly, there was no evidence of cellular damage from the magnetic force. This study also highlighted the flexibility of magnetic patterning in 2D and 3D cell cultures to pattern cell aggregates with different shapes and sizes, which can be created with relative ease.While some studies have demonstrated limited cytotoxicity when using magnetic labeling of cells, there is a still high risk that more subtle cellular functions will be affected when relying on the endocytosis of MNPs to render cells responsive to magnetic stimulus. To avoid this, some studies have sought to limit MNP association to the cell membrane, rather than promoting internalization. This avoids any interference MNPs may have with cell machinery and achieves a similar degree of cell maneuverability by an external magnetic field. Dzamukova et al.7373. M. R. Dzamukova, E. A. Naumenko, E. V. Rozhina, A. A. Trifonov, and R. F. Fakhrullin, Nano Res. 8, 2515 (2015) deposited cationic polyelectrolyte-coated MNPs directly onto the membrane of human skin fibroblasts creating a semipermeable mesoporous layer that was magnetically responsive. The labeled cells responded to a magnetic field, aligning themselves to the location of highest field strength within the culture wells. This was done in a layered fashion to create bilayered, round, multicellular cultures or could also be adapted to form cell spheroids. A thorough analysis demonstrated that these MNPs did not inhibit enzymatic pathways, membrane or cell integrity, proliferation, and cytoskeleton formation and did not induce apoptotic pathways in either primary or cancer cells. In addition to the benefits this technique brings in potentially reducing cytotoxicity, it was also noted that this simple method could label cells with MNPs in just minutes under cell-friendly conditions.Another way to minimize the potential for cytotoxicity and altered cell function is to incorporate MNPs into the ECM of a cell aggregate rather than within an individual cell or its membrane. Bratt-Leal et al.7474. A. M. Bratt-Leal, K. L. Kepple, R. L. Carpenedo, M. T. Cooke, and T. C. McDevitt, Integr. Biol. 3, 1224 (2011). https://doi.org/10.1039/c1ib00064k trapped magnetic microparticles (Mag-MPs) within the ECM of stem cell spheroids as they formed. Their work confirmed that the MNPs were not endocytosed by the cells, but the cell aggregate could still be moved across multiple length scales (from few micrometers to millimeters) upon application of an external magnetic field. Experiments under static conditions demonstrated excellent maneuverability, where spheroid aggregates could be translated, rotated, and allocated to desired positions based on the relative location and direction of the external magnetic field, with spheroids contained a greater proportion of Mag-MPs being more sensitive to the applied magnetic field. However, the same level of control was not observed under hydrodynamic testing, which aimed to mimic the conditions found in bioreactor cell cultures for biomanufacturing. Although the cell aggregates did respond to the magnetic forces under dynamic conditions, the shear forces within the culture medium made it difficult to control the spheroids with great accuracy.Negative magnetophoresis may present a suitable alternative to overcome the shortcomings of positive magnetophoresis methods. Here, cells are forced by the surrounding medium into a location of lowest field strength. Cytotoxicity risks are minimal, as MNPs do not need to be internalized or associated with the cell membrane and only the medium in which the cells are need to be magnetic. Furthermore, if using a paramagnetic medium, the manipulation is temporary, and cells are largely undisturbed. Akiyama and Morishima6969. Y. Akiyama and K. Morishima, Appl. Phys. Lett. 98, 163702 (2011). https://doi.org/10.1063/1.3581883 used negative magnetophoresis to shuttle human T cells into an area of lowest magnetic field strength and assemble an “egg” shape that changed to spheroid over a few days [Fig. 3(d)]. Using gadolinium diethylenetriaminepentaacetic acid as a paramagnetic medium, the cells were forced to move to the destination of interest by the movement of the solution around them. This enabled a labelfree, rapid, and easy to use method of 3D cell patterning. In an alternative application, Akiyama et al.6666. H. Akiyama, A. Ito, Y. Kawabe, and M. Kamihira, J. Biomed. Mater. Res. Part A 92A, 1123 (2009). https://doi.org/10.1002/jbm.a.32313 used PEG-modified magnetite particles (PEG-Mags) to pattern a 3D coculture of mouse myoblast/fibroblast and human keratinocyte cells by controlling deposition of PEG-Mags onto the surface of a culture dish, producing a patterned nonadherent area. Human keratinocyte cells attached only to the areas without PEG-Mags. When the PEG-Mags were removed, mouse myoblast cells were seeded onto the remaining area, generating a specific pattern of both cell types.

Overall, magnetophoresis is a valuable tool in cell patterning as cells can be organized remotely, with considerable ease and high specificity. However, methods involving magnetic labeling may introduce cytotoxic effects to cells or interrupt their signaling, ultimately jeopardizing the utility of these cells. The hope is that future research can expand upon the collection of techniques that rely on negative magnetophoresis or a harmless integration of magnetic particles with cells instead.

3. Acoustic cell patterning

Acoustic forces have gained increasing popularity in recent years as a means to precisely modulate the in situ positioning of biological matter. In this process, acoustic forces are applied to a conductive medium, resulting in the formation of periodically fluctuating pressure fields. The formation of these fluctuating fields results in the propagation of time-averaged forces, which are able to push suspended matter toward acoustic nodes/antinodes. The precise size of these nodes can be controlled by modulating the acoustic wavelength, with higher range frequencies facilitating decreased node sizes, and thus, more precise manipulation of smaller particles due to the internodal spacing produced being on the same size scale as a single cell [Figs. 4(a)–4(d)].Initial application of acoustic forces to modulate cellular positioning in 2D demonstrated success with large scale manipulation, such as with the positioning of cell aggregates.76,7776. Z. Tian et al., Sci. Adv. 5, eaau6062 (2019). https://doi.org/10.1126/sciadv.aau606277. B. Vanherberghen et al., Lab Chip 10, 2727 (2010). https://doi.org/10.1039/c004707d However, more recent endeavors have sought to increase the resolution of cellular manipulation from large-scale aggregates to the single cell scale, where precise single cell positioning has now been demonstrated.7878. Z. Ma et al., Adv. Mater. 32, 1904181 (2020). https://doi.org/10.1002/adma.201904181 For example, Collins et al. demonstrated a method for active patterning of individual cells in an acoustic field defined in two dimensions.7979. D. J. Collins, B. Morahan, J. Garcia-Bustos, C. Doerig, M. Plebanski, and A. Neild, Nat. Commun. 6, 8686 (2015). https://doi.org/10.1038/ncomms9686 Selective cell isolation and positioning in a high-throughput manner was achieved using high-frequency acoustics (100–200 MHz) and could facilitate single cell image analysis in situ. Tian et al. further extended this work through the application of wave number-spiral acoustic tweezers, capable of dynamically reshaping surface acoustic wave wavefields across 2D substrates75,7675. S. Oberti, A. Neild, and J. Dual, J. Acoust. Soc. Am. 121, 778 (2007). https://doi.org/10.1121/1.240492076. Z. Tian et al., Sci. Adv. 5, eaau6062 (2019). https://doi.org/10.1126/sciadv.aau6062 [Figures 4(e)–4(j)]. The ability to modulate these pressure fields in real-time enabled dynamic and programmable particle/cell manipulation, as demonstrated by controlling cell positioning, grouping, and rotation to more precisely control cell behavior.More recently, efforts have been made to modulate cellular positioning within 3D cultures using acoustic forces. For example, Koo et al. paired direct extrusion of sodium alginate hydrogels with standing acoustic waves to align fibroblast cells within an extruded bio-ink.8080. K. Koo, A. Lenshof, L. T. Huong, and T. Laurell, Micromachines 12, 3 (2020). https://doi.org/10.3390/mi12010003 Interestingly, fibroblast distribution could be patterned toward the center of the extruded hydrogel strands or used to quickly and efficiently align the fibroblasts in either single or quadruple streams via the actuation of acoustic frequencies between 2 and 4 MHz, with the precise distribution being dependent on the frequency applied. It was also demonstrated that direct cell connections were retained during switching between single- and multicell streams, thus demonstrating potential application in the development of more complex biological structures such as vascularized networks, which require retained cellular contact to regulate cellular function.Acoustic forces offer significant advantages over other noncontact techniques for cellular positioning, such as its demonstrated biocompatibility across a range of frequencies and ability to avoid photobleaching/local heat stress, often associated with optical trapping and electrical fields.81–8481. X. Ding et al., Proc. Natl. Acad. Sci. U.S.A. 109, 11105 (2012). https://doi.org/10.1073/pnas.120928810982. F. Guo et al., Proc. Natl. Acad. Sci. U.S.A. 112, 43 (2015). https://doi.org/10.1073/pnas.142206811283. A. G. Guex, N. Di Marzio, D. Eglin, M. Alini, and T. Serra, Mater. Today Bio. 10, 100110 (2021). https://doi.org/10.1016/j.mtbio.2021.10011084. Y. Gao, A. K. Fajrial, T. Yang, and X. Ding, Biomater. Sci. 9, 1574 (2021). https://doi.org/10.1039/D0BM01269F However, the forces generated by these acoustic cell patterning systems are relatively weak, meaning that the transition from closed 2D systems to more in vivo like scenarios can be readily disrupted by environmental factors such as mechanical agitation and shear stresses.8383. A. G. Guex, N. Di Marzio, D. Eglin, M. Alini, and T. Serra, Mater. Today Bio. 10, 100110 (2021). https://doi.org/10.1016/j.mtbio.2021.100110 It is also important to note that acoustic forces also affect cell properties in ways that are not yet well understood. For example, Citsabehsan et al. showed that acoustic forces affect cellular metabolic activity, adhesion speed, and proliferation rate,8585. C. Devendran, J. Carthew, J. E. Frith, and A. Neild, Adv. Sci. 6, 1902326 (2019). https://doi.org/10.1002/advs.201902326 and Ambattu et al. have shown a direct role for acoustic forces in regulating mesenchymal stromal cell (MSC) osteogenic differentiation.8686. L. A. Ambattu, A. Gelmi, and L. Y. Yeo, Small 18, 2106823 (2022). https://doi.org/10.1002/smll.202106823 While this means that care must be taken to understand what effects acoustic forces have on a cell population, it also exposes new opportunities to use acoustic forces to modify cell fate. However, future efforts must seek to untangle the effects of acoustics on cell positioning from these more direct effects if this is to effectively direct cell fate.Using acoustics to control the distribution of biochemical factors, rather than cells themselves, may bypass any potential influences of direct acoustic forces on cellular behaviors. An advantage of this is that it can significantly reduce the acoustic exposure times experienced by the cells, while simultaneously directing cellular migration, positioning, and subsequent development via spatiotemporal control of growth factor distribution. One limitation is that the forces required for precise patterning are proportional to the object volume, providing a significant challenge for nanoscaled patterning of biochemical factors and matrix fibers. As a result, several groups have aimed to bypass this hurdle through the development of two-stage systems designed to direct growth factor positioning without the need for significant reductions in the acoustic wavelength. Shanjani et al., for example, demonstrated an acoustic droplet ejection technique to control recombinant bone morphogenetic protein-2 (rhBMP-2) distribution across polycaprolactone (PCL)-based tissue engineering constructs.8787. Y. Shanjani, S. M. Siebert, D. F. E. Ker, A. E. Mercado-Pagán, and Y. P. Yang, Tissue Eng. Part A 26, 602 (2020). https://doi.org/10.1089/ten.tea.2019.0271 This study demonstrated high-resolution distribution of BMP-2 spots across the 3D growth substrate (250 μm spots with 700 μm center-to-center spacing), which directly correlated to the spatial alkaline phosphatase staining of C2C12 mouse muscle cells. Lu et al. further advanced these findings with an acoustically responsive scaffold in which release of a GF was noninvasively and spatiotemporally controlled using focused ultrasound.8888. X. Lu et al., Acta Biomater. 113, 217 (2020). https://doi.org/10.1016/j.actbio.2020.06.015 Here, a fibrin matrix was combined with a growth factor loaded phase-shift emulsion, which upon acoustic excitation, released its cargo to the surrounding medium via acoustic droplet vaporization, while the surrounding fibrin matrix remained unchanged. Fibroblast migration toward areas of GF release was significantly increased, thus demonstrating indirect control of 3D cellular positioning using acoustic forces.

While these findings show encouraging results in using acoustics for cell patterning, there remains a significant caveat, being that our current understanding of how cells respond to these acoustic forces remains limited. When combined with technical complexity of most current acoustic systems, it becomes difficult to use these more widely to develop fundamental understanding or translation to a clinical setting. However, with future improvements on both technological and biological aspects, acoustics may open up novel avenues toward therapeutic applications via effects on cellular positioning and possibly even targeted effects on signaling pathways to drive cell behavior and lineage development.

4. Cell origami

Cell origami is one of many technologies that seek to mimic nature to create and repair tissues. Taking inspiration from the Japanese art of paper folding to manufacture 3D cellular structures,8989. B. Kresling, MRS Proc. 1420, 42 (2012). https://doi.org/10.1557/opl.2012.536 cellular origami could be used to mimic some of the natural folding processes that occur during tissue development, such as epithelial invagination where epithelial cells in 2D sheets undergo self-bending into ridges, folds, pits, and tubes9090. E. J. Pearl, J. Li, and J. B. A. Green, Philos. Trans. R. Soc. B 372, 20150526 (2017). https://doi.org/10.1098/rstb.2015.0526 (Fig. 5). Key processes where folding is relevant include neurulation where the neural plate becomes the neural tube,9292. N. D. E. Greene and A. J. Copp, Prenat. Diagn. 29, 303 (2009). https://doi.org/10.1002/pd.2206 gut morphogenesis,9393. T. Savin et al., Nature 476, 57 (2011). https://doi.org/10.1038/nature10277 and even cortical folding during brain development.9494. K. E. Garcia, C. D. Kroenke, and P. V. Bayly, Philos. Trans. R. Soc. B 373, 20170321 (2018). https://doi.org/10.1098/rstb.2017.0321 Cell origami relies upon the fact that cells apply traction forces to the substrates that they adhere to, where the forces applied by cells upon microsized constructs initiate folding to create a 3D structure.9595. K. Kuribayashi-Shigetomi, H. Onoe, and S. Takeuchi, PLoS One 7, e51085 (2012). https://doi.org/10.1371/journal.pone.0051085 A successful cell origami strategy must, therefore, balance the properties of the biomaterials with the strength and patterning of forces applied by the cells. Typically, this is augmented by the provision of some sort of stimulus to initiate folding where temperature and electrical and chemical signals have all been used to initiate a change in the surface tension or shrinkage of a biomaterial and subsequently facilitate folding of the structure in the direction of forces applied by the attached cells95,9695. K. Kuribayashi-Shigetomi, H. Onoe, and S. Takeuchi, PLoS One 7, e51085 (2012). https://doi.org/10.1371/journal.pone.005108596. V. A. Bolaños Quiñones, H. Zhu, A. A. Solovev, Y. Mei, and D. H. Gracias, Adv. Biosyst. 2, 1800230 (2018). https://doi.org/10.1002/adbi.201800230Takeuchi et al. were the first group to develop origami systems for cell/tissue patterning purposes and produced regular tetragon, regular dodecahedron, and cylindrical helical tubes.9595. K. Kuribayashi-Shigetomi, H. Onoe, and S. Takeuchi, PLoS One 7, e51085 (2012). https://doi.org/10.1371/journal.pone.0051085 Continuing with this approach, He et al. implemented origami self-folding to pattern cocultured cells within 3D microstructures. NIH/3T3 cells were first coated onto unfolded origami baseplates before HepG2 hepatocytes were seeded on top. Upon addition of alginate lysase, a sacrificial alginate layer in the baseplates was removed, triggering folding of the 3T3 cells into a dodecahedral microstructure around the hepatocytes.9191. Q. He, T. Okajima, H. Onoe, A. Subagyo, K. Sueoka, and K. Kuribayashi-Shigetomi, Sci. Rep. 8, 4556 (2018). https://doi.org/10.1038/s41598-018-22598-x More recently, Ge et al. have used DNA origami nanostructures to control the assembly of individual cells into origami clusters.