Single-Cell Screening through Cell Encapsulation in Photopolymerized Gelatin Methacryloyl

: This study evaluated the potential of gelatin methacryloyl (GelMA) for single-cell screening compared to polyethylene glycol diacrylate (PEGDA). GelMA photopolymerized at 1000–2000 mJ/cm 2 produced consistent patterns and supported HeLa cell viability. GelMA (5% w/v) facilitated better cell collection within 2 days due to its shape retention. GelMA demonstrated biocompatibility with HeLa cells exhibiting exponential proliferation and biodegradation over 5 days. The average cell displacement over 2 days was 16 µ m. Two targeted cell recovery strategies using trypsin were developed: one for adherent cells encapsulated at 800 mJ/cm 2 , and another for floating cells encapsulated at 800 mJ/cm 2 , enabling the selective removal of unwanted cells. These findings suggest GelMA as a promising biomaterial for single-cell screening applications, offering advantages over PEGDA in cell encapsulation and targeted recovery.


Introduction
Single-cell screening is a crucial technique for collecting specific cells based on the desired characteristics [1][2][3].A major method for obtaining target cell populations is flow cytometric cell sorting [4,5], which is based on the optical and fluorescent characteristics of individual cells [6,7].During flow cytometry, suspended cells pass through a detector in a short period of time.It is important to note that flow cytometry has inherently limited measurement quality.
Image-based single-cell screening systems overcome these limitations by utilizing high-resolution imaging and temporal analysis, allowing for the detection of weak signals and transient events [8].This approach offers flexible cell collection methods, including single-pipette [9,10] and laser-based techniques [11].Collected cells, both of interest [12][13][14] or not [15], can be encapsulated in photopolymerizable hydrogels like polyethylene glycol diacrylate (PEGDA) using light irradiation.Although the number of target cells is often greater than unwanted cells, and encapsulating cells of interest in PEGDA hydrogels is relatively straightforward, encapsulating negative cells is less efficient.While PEGDA hydrogels are suitable for DNA extraction, they have limitations such as slow degradation [16] and cytotoxicity at lower molecular weights [17,18].For viable cell recovery and subsequent analysis, the surrounding hydrogel needs to degrade.
Gelatin methacrylate (GelMA) offers distinct advantages.This biocompatible material degrades by both cells and enzymes [19][20][21], making it more suitable for cell screening via encapsulation than PEGDA.The long-term viability of cells encapsulated in GelMA micromodules [22] and microspheres [23,24] has been established.Cells were acoustically patterned into diverse arrangements and encapsulated in GelMA for applications such as studying stem cell differentiation [25], constructing heterogeneous tissue models [26], and high-throughput drug testing [27].GelMA hydrogels can be degraded using readily available enzymes like trypsin [28] and collagenase [29,30], facilitating efficient cell collection.While we have developed an automated single-cell encapsulation system [31], the specific properties of GelMA in the context of single-cell screening and the efficient collection of target cells after encapsulation remain unexplored.
To address this gap, we investigated the effects of GelMA hydrogels on cell proliferation, biodegradation, and cell motility within the gels for target single-cell collection.We tested both suspended and adherent cells for collecting cells encapsulated in GelMA.Our study successfully demonstrates the recovery of single floating and adherent cells through gel lysis using a time-controlled trypsin treatment.
HeLa cells (RCB0007, RIKEN BRC, Wako, Japan) were cultured in minimum essential media (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillinstreptomycin (Pen/Strep).The culture medium was removed, and cells were washed with 3 mL of PBS.Subsequently, 3 mL of 0.25% trypsin-EDTA 1X (Gibco TM ., 25200072, USA) was added to the dish and incubated for 4 min in a CO 2 incubator.Following incubation, 3 mL of the medium was added to the trypsinized cells, and the suspension was centrifuged for 3 min to remove the supernatant.Finally, the cells were resuspended for subsequent use in either a culture medium or bioink.
The GelMA bioink was photopolymerized using our DMD-based photoirradiation system, as described previously [31,34].For cell encapsulation, the GelMA hydrogel and cells were exposed to 405 nm light at 100-300 mW/cm 2 for an integrated light dose of 600-2000 mJ/cm 2 .The irradiation time ranged from 2 s to 8 s.The light pattern consisted of an arrangement of four squares, each measuring 98 µm.The ambient temperature was maintained at 23 • C throughout the process.

Live/Dead Assay
Calcein-AM (C396, Dojindo, Tokyo, Japan) (2.0 µM) and 4.0 µM propidium (PI, 341-07881, Dojindo) were prepared in PBS(-).Cell viability was assessed immed after gel polymerization through light irradiation.Cells were washed with PBS( transferred to a 50 mm Falcon petri dish.Subsequently, 100 µL of the cell staining so was added to the cells in the dish.To allow for stain diffusion through the gel, th was incubated for 35 min at 37 °C and 5% CO2 in an incubator.For comparison, suspension without photopolymerized gel was also incubated for 15 min as the ne control. After incubation, the cells were examined under a microscope (Eclipse Ti-U, N Tokyo, Japan) equipped with a cooled camera (Nikon, DIGITAL SIGHT, DS-Qi1M kyo, Japan) using fluorescent filters (B2A: 150 ms, C-FL-C TRITC: 150 ms).Live cel played green fluorescence, while dead cells emitted red fluorescence.Cell viabilit calculated using the following equation: Cell viability (%) = NLive/(NLive + NDead), wher is number of live cells and NDead is number of dead cells.

Observation of Cells Encapsualted in GelMA
HeLa cells were encapsulated in GelMA at 1000 mJ/cm 2 and were washed wit and cultured in a 50 mm petri dish containing the conditioned medium (MEM su mented with 10% FBS and 1% Pen/Strep).The dish was kept in an incubator at 37 ° 5% CO2, and observed every day.The encapsulated cells were characterized to ob the cell proliferation, biodegradation of the gel, and cell motility over time.The biod dability of GelMA was investigated by comparing the gel area after encapsulation b irradiation.Gels with and without cells were measured for 5 days.

Encapsulation of Attached and Suspended Cells in GelMA
HeLa cells were encapsulated in GelMA using two strategies: attachment to verslip or suspension in a cuvette.Both methods utilized a 40 µm × 40 µm square p for encapsulation.Cells were (1) attached to a coverslip (Figure 1c) or (2) suspende cuvette (Figure 1h).These cells were encapsulated in GelMA with a 40 µm × 40 µm s pattern.We needed more time to detach adherent cells as the GelMA hydrogels were

Live/Dead Assay
Calcein-AM (C396, Dojindo, Tokyo, Japan) (2.0 µM) and 4.0 µM propidium iodide (PI, 341-07881, Dojindo) were prepared in PBS(-).Cell viability was assessed immediately after gel polymerization through light irradiation.Cells were washed with PBS(-) and transferred to a 50 mm Falcon petri dish.Subsequently, 100 µL of the cell staining solution was added to the cells in the dish.To allow for stain diffusion through the gel, the dish was incubated for 35 min at 37 • C and 5% CO 2 in an incubator.For comparison, a cell suspension without photopolymerized gel was also incubated for 15 min as the negative control.
After incubation, the cells were examined under a microscope (Eclipse Ti-U, Nikon, Tokyo, Japan) equipped with a cooled camera (Nikon, DIGITAL SIGHT, DS-Qi1Mc, Tokyo, Japan) using fluorescent filters (B2A: 150 ms, C-FL-C TRITC: 150 ms).Live cells displayed green fluorescence, while dead cells emitted red fluorescence.Cell viability was calculated using the following equation: Cell viability (%) = N Live /(N Live + N Dead ), where N Live is number of live cells and N Dead is number of dead cells.

Observation of Cells Encapsualted in GelMA
HeLa cells were encapsulated in GelMA at 1000 mJ/cm 2 and were washed with PBS and cultured in a 50 mm petri dish containing the conditioned medium (MEM supplemented with 10% FBS and 1% Pen/Strep).The dish was kept in an incubator at 37 • C and 5% CO 2 , and observed every day.The encapsulated cells were characterized to observe the cell proliferation, biodegradation of the gel, and cell motility over time.The biodegradability of GelMA was investigated by comparing the gel area after encapsulation by light irradiation.Gels with and without cells were measured for 5 days.

Encapsulation of Attached and Suspended Cells in GelMA
HeLa cells were encapsulated in GelMA using two strategies: attachment to a coverslip or suspension in a cuvette.Both methods utilized a 40 µm × 40 µm square pattern for encapsulation.Cells were (1) attached to a coverslip (Figure 1c) or (2) suspended in a cuvette (Figure 1h).These cells were encapsulated in GelMA with a 40 µm × 40 µm square pattern.We needed more time to detach adherent cells as the GelMA hydrogels were more crosslinked, making degradation take longer.To collect floating cells encapsulated in GelMA hydrogels, we can ignore cell dissociation and focus on the rapid degradation of GelMA hydrogels with a lower light integral dose of 600 mJ/cm 2 .
(1) A suspension of HeLa cells was incubated for 2 h in a CO 2 incubator at 37 • C to promote attachment (Figure 1c).A cuvette was made and 20 µL of bioink was injected (Figure 1d).The cuvette with the bioink was placed on the stage of an inverted microscope (Nikon ECLIPSE Ti-U, Tokyo, Japan).HeLa cells were encapsulated on the coverslip within the bioink using 405 nm light with an integral dose of 800 mJ/cm 2 (Figure 1e).( 2) HeLa cells were suspended in 2 mL of bioink (Figure 1h).The final concentration of cells was 4 × 10 6 cells/mL.Single HeLa cells were encapsulated on the coverslip by irradiating the bioink with 405 nm at a light integral of 600 mJ/cm 2 (Figure 1i).

Collection of Cells with Trypsin
The time-controlled application of trypsin facilitated the selective recovery of encapsulated cells (Figure 1f,g, or Figure 1k,l).The coverslip with encapsulated cells was transferred to a 50 mm Petri dish and rinsed with PBS(-) to remove non-encapsulated cells.Next, trypsin (Gibco Ltd., 0.25% Trypsin-EDTA (1X)) was added to detach adherent cells and degrade the GelMA hydrogel.The exposed cells were detached first, followed by the collection of encapsulated cells after the dissolution of the gel.The dissolution and cell release were monitored using an inverted microscope and a cooled camera.

Basic Characterization with GelMA
Figure 2a-e presents the top view of a GelMA hydrogel containing HeLa cells which were irradiated at 1070 mJ/cm 2 to 1920 mJ/cm 2 .The gel was transparent after polymerization of GelMA at a concentration of 5%w/v, and the cells encapsulated in the gel were visible.Encapsulating the cells in a thin layer of gel helped maintain their immobilization and viability.Figure 2f depicts the individual gel units, each measuring approximately 92-94 µm after photopolymerization with 98 µm square light patterns.In single cell screening, the size of photocured hydrogel is important to determine the limits of the inter space between two patterns.If the size of the pattern is too large or too small compared to the designed values, some margins need to be considered.The data show that within the range of this light integral, the size of the formed hydrogel does not change significantly.
Micro 2024, 4, FOR PEER REVIEW 4 crosslinked, making degradation take longer.To collect floating cells encapsulated in GelMA hydrogels, we can ignore cell dissociation and focus on the rapid degradation of GelMA hydrogels with a lower light integral dose of 600 mJ/cm 2 .
(1) A suspension of HeLa cells was incubated for 2 h in a CO2 incubator at 37 °C to promote attachment (Figure 1c).A cuvette was made and 20 µL of bioink was injected (Figure 1d).The cuvette with the bioink was placed on the stage of an inverted microscope (Nikon ECLIPSE Ti-U, Tokyo, Japan).HeLa cells were encapsulated on the coverslip within the bioink using 405 nm light with an integral dose of 800 mJ/cm 2 (Figure 1e).( 2) HeLa cells were suspended in 2 mL of bioink (Figure 1h).The final concentration of cells was 4 × 10 6 cells/mL.Single HeLa cells were encapsulated on the coverslip by irradiating the bioink with 405 nm at a light integral of 600 mJ/cm 2 (Figure 1i).

Collection of Cells with Trypsin
The time-controlled application of trypsin facilitated the selective recovery of encapsulated cells (Figure 1f,g, or Figure 1k,l).The coverslip with encapsulated cells was transferred to a 50 mm Petri dish and rinsed with PBS(-) to remove non-encapsulated cells.Next, trypsin (Gibco Ltd., 0.25% Trypsin-EDTA (1X)) was added to detach adherent cells and degrade the GelMA hydrogel.The exposed cells were detached first, followed by the collection of encapsulated cells after the dissolution of the gel.The dissolution and cell release were monitored using an inverted microscope and a cooled camera.

Basic Characterization with GelMA
Figure 2a-e presents the top view of a GelMA hydrogel containing HeLa cells which were irradiated at 1070 mJ/cm 2 to 1920 mJ/cm 2 .The gel was transparent after polymerization of GelMA at a concentration of 5%w/v, and the cells encapsulated in the gel were visible.Encapsulating the cells in a thin layer of gel helped maintain their immobilization and viability.Figure 2f depicts the individual gel units, each measuring approximately 92-94 µm after photopolymerization with 98 µm square light patterns.In single cell screening, the size of photocured hydrogel is important to determine the limits of the inter space between two patterns.If the size of the pattern is too large or too small compared to the designed values, some margins need to be considered.The data show that within the range of this light integral, the size of the formed hydrogel does not change significantly.Photopolymerized 5%w/v GelMA with encapsulated cells showed high viability of 96% (43/45 cells).Ten sites were encapsulated with 1200 mJ/cm 2 light irradiation, and viability was assessed immediately after fluorescence microscopy using Calcein-AM and PI staining.The negative control reached 98% (173/177 cells).Figure 3 shows the microscopic views of cells under different conditions.Figure 3a-c depict cells encapsulated in a 5%w/v GelMA hydrogel that were photopolymerized at 1200 mJ/cm 2 .Figure 3a is a bright field image showing the overall cell population.Figure 3b shows the green fluorescence of livecell stained with Calcein-AM, indicating viable cells.Figure 3c looks black, suggesting that no dead cells were detected with red fluorescence, stained with PI. Figure 3d-f  Photopolymerized 5%w/v GelMA with encapsulated cells showed high viability of 96% (43/45 cells).Ten sites were encapsulated with 1200 mJ/cm 2 light irradiation, and viability was assessed immediately after fluorescence microscopy using Calcein-AM and PI staining.The negative control reached 98% (173/177 cells).Figure 3 shows the microscopic views of cells under different conditions.Figure 3a-c depict cells encapsulated in a 5%w/v GelMA hydrogel that were photopolymerized at 1200 mJ/cm 2 .Figure 3a is a bright field image showing the overall cell population.Figure 3b shows the green fluorescence of livecell stained with Calcein-AM, indicating viable cells.Figure 3c looks black, suggesting that no dead cells were detected with red fluorescence, stained with PI. Figure 3d-f represents the negative control conditions without any treatment.

Biodegradability of GelMA
Figure 4a,b demonstrates the biodegradation of GelMA hydrogels with and without encapsulated cells after 5 days.The gel containing cells exhibited significant degradation, while the cell-free gel remained largely intact.While the cell-free gel showed a slight decrease in the area, it was not statistically significant.
The change in the gel area over time was quantified to assess cell biodegradability (Figure 4c).A 100 µm × 100 µm square pattern was used for light irradiation.The initial cell count, and the initial area of the cell-free gel are shown in the graph.The graph highlights a significant difference in the area between the initial cell-embedded gels and the cell-free gel.The rate of decrease in the area is related to time and is not strongly related to the initial cell count.This reason is explained by the fact that the number of cells in the hydrogels is comparable on Day 5. A significant decrease in the gel area occurred after Day 3, suggesting cell-mediated degradation.The area of the cell-free gel stabilized at approximately 10 × 10 3 µm 2 by Day 5, while the area of the initial cell-embedded gels, averaged across four hydrogels, decreased to approximately 3.4 ± 0.2 × 10 3 µm 2 .
Figure 4d examines cell proliferation within 100 µm × 100 µm GelMA hydrogels after immobilization.Series 2-5 in the graph represent the initial number of encapsulated cells on Day 0. The cells within the gel continued to proliferate throughout the 5 days, reaching several dozen cells under all conditions by Day 5.The exponential growth curve observed resembles normal cell proliferation patterns [35].

Behavior of Cells Encapsulated in the GelMA Hydrogel
Figure 5a,b, respectively, presents observations of HeLa cells encapsulated in the GelMA hydrogel over 2 days with initial cell number of 3 and 5.In Figure 5a, a cell in the upper left corner exemplifies cell division occurring between Day 0 and Day 1, while the lower right corner shows cell division from Day 1 to Day 2. The hydrogel appears slightly The change in the gel area over time was quantified to assess cell biodegradability (Figure 4c).A 100 µm × 100 µm square pattern was used for light irradiation.The initial cell count, and the initial area of the cell-free gel are shown in the graph.The graph highlights a significant difference in the area between the initial cell-embedded gels and the cell-free gel.The rate of decrease in the area is related to time and is not strongly related to the initial cell count.This reason is explained by the fact that the number of cells in the hydrogels is comparable on Day 5. A significant decrease in the gel area occurred after Day 3, suggesting cell-mediated degradation.The area of the cell-free gel stabilized at approximately 10 × 10 3 µm 2 by Day 5, while the area of the initial cell-embedded gels, averaged across four hydrogels, decreased to approximately 3.4 ± 0.2 × 10 3 µm 2 .
Figure 4d examines cell proliferation within 100 µm × 100 µm GelMA hydrogels after immobilization.Series 2-5 in the graph represent the initial number of encapsulated cells on Day 0. The cells within the gel continued to proliferate throughout the 5 days, reaching several dozen cells under all conditions by Day 5.The exponential growth curve observed resembles normal cell proliferation patterns [35].Figure 6a-d depicts a graph tracing the coordinates of the hydrogel corners and capsulated cells, with the origin at a grid intersection.Cells remained confined withi gel, even when encountering the edge, and tended to move inward.Figure 6e show measured displacement of cells from their initial positions.The average cell migration 12.2 µm ± 7.6 µm (N = 14) on Day 1 and 16.1 ± 9.6 µm (N = 14) on Day 2. A stiffer Ge hydrogel might potentially reduce cell displacement, as reported in [36].

Recovery of Cells Encapsulated in GelMA
This study presents a novel technique for recovering adherent cells using a blue laser and DMD to photopolymerize a gel around individual cells.Patterned irradiation at 800 mJ/cm 2 selectively encapsulated HeLa cells in the GelMA hydrogel on a coverslip (Figure 7a).A subsequent 4 min trypsin treatment effectively removed unwanted cells, leaving only the encapsulated HeLa cells (Figure 7b).While longer trypsin exposure (10 min) did not fully degrade the gel, the desired cells remained encapsulated (Figure 7c).This method allows for the detachment of unwanted cells while specifically collecting the targeted cells.For suspended cells, irradiation at 600 mJ/cm 2 resulted in HeLa cell encapsulation, but these cells were not readily detached by rinsing (Figure 8).Rinsing removed unwanted cells, and the encapsulated cells were subsequently collected by degrading the GelMA with trypsin.Video S1 further illustrates the gel dissolution process with trypsin and the release of cells from the GelMA hydrogels after 5 min of treatment.

Recovery of Cells Encapsulated in GelMA
This study presents a novel technique for recovering adherent cells using a blue laser and DMD to photopolymerize a gel around individual cells.Patterned irradiation at 800 mJ/cm 2 selectively encapsulated HeLa cells in the GelMA hydrogel on a coverslip (Figure 7a).A subsequent 4 min trypsin treatment effectively removed unwanted cells, leaving only the encapsulated HeLa cells (Figure 7b).While longer trypsin exposure (10 min) did not fully degrade the gel, the desired cells remained encapsulated (Figure 7c).This method allows for the detachment of unwanted cells while specifically collecting the targeted cells.

Recovery of Cells Encapsulated in GelMA
This study presents a novel technique for recovering adherent cells using a blue laser and DMD to photopolymerize a gel around individual cells.Patterned irradiation at 800 mJ/cm 2 selectively encapsulated HeLa cells in the GelMA hydrogel on a coverslip (Figure 7a).A subsequent 4 min trypsin treatment effectively removed unwanted cells, leaving only the encapsulated HeLa cells (Figure 7b).While longer trypsin exposure (10 min) did not fully degrade the gel, the desired cells remained encapsulated (Figure 7c).This method allows for the detachment of unwanted cells while specifically collecting the targeted cells.For suspended cells, irradiation at 600 mJ/cm 2 resulted in HeLa cell encapsulation, but these cells were not readily detached by rinsing (Figure 8).Rinsing removed unwanted cells, and the encapsulated cells were subsequently collected by degrading the GelMA with trypsin.Video S1 further illustrates the gel dissolution process with trypsin and the release of cells from the GelMA hydrogels after 5 min of treatment.For suspended cells, irradiation at 600 mJ/cm 2 resulted in HeLa cell encapsulation, but these cells were not readily detached by rinsing (Figure 8).Rinsing removed unwanted cells, and the encapsulated cells were subsequently collected by degrading the GelMA with trypsin.Video S1 further illustrates the gel dissolution process with trypsin and the release of cells from the GelMA hydrogels after 5 min of treatment.mild enzymatic conditions.The hydrolytic degradation of PEGDA is a slow process compared to the enzymatic degradation of GelMA.Our approach takes advantage of the different properties inside and outside the GelMA hydrogels for targeted cell collection.Our results confirm that prolonged exposure to light increases the dissolution time of GelMA hydrogels, which is consistent with previous reports on the proteolytic degradation of GelMA hydrogels [28,29].

Conclusions
This study highlights the suitability of GelMA over PEGDA for cell encapsulation.The dimension and viability evaluations of GelMA photopolymerized with an integrated light intensity of 1000-2000 mJ/cm 2 showed similar pattern formation.Interestingly, HeLa cells encapsulated in 5%w/v GelMA were better collected within a 2-day culture period due to GelMA's shape stability.Further demonstrating the biocompatibility of GelMA, HeLa cells within the gel exhibited exponential proliferation over 5 days.The gel area where cells were encapsulated significantly decreased after 5 days, suggesting biodegradation by the cells.The averaged cell displacement remained less than 20 µm from the initial position.
We demonstrated two targeted cell recovery strategies.(1) Adherent cells: Targeted adherent cells were fixed on the coverslip using 800 mJ/cm 2  This study chose GelMA over PEGDA due to its superior degradability with trypsin, as the degradation of PEGDA is more difficult [12][13][14].PEGDA hydrogels primarily degrade via hydrolysis of the ester bonds, with degradation occurring more rapidly at an acidic or alkaline pH [37].This property of PEGDA has made it challenging to collect live cells.In contrast, GelMA hydrogels degraded more readily than PEGDA hydrogels under mild enzymatic conditions.The hydrolytic degradation of PEGDA is a slow process compared to the enzymatic degradation of GelMA.Our approach takes advantage of the different properties inside and outside the GelMA hydrogels for targeted cell collection.Our results confirm that prolonged exposure to light increases the dissolution time of GelMA hydrogels, which is consistent with previous reports on the proteolytic degradation of GelMA hydrogels [28,29].

Conclusions
This study highlights the suitability of GelMA over PEGDA for cell encapsulation.The dimension and viability evaluations of GelMA photopolymerized with an integrated light intensity of 1000-2000 mJ/cm 2 showed similar pattern formation.Interestingly, HeLa cells encapsulated in 5%w/v GelMA were better collected within a 2-day culture period due to GelMA's shape stability.Further demonstrating the biocompatibility of GelMA, HeLa cells within the gel exhibited exponential proliferation over 5 days.The gel area where cells were encapsulated significantly decreased after 5 days, suggesting biodegradation by the cells.The averaged cell displacement remained less than 20 µm from the initial position.
We demonstrated two targeted cell recovery strategies.(1) Adherent cells: Targeted adherent cells were fixed on the coverslip using 800 mJ/cm 2 light irradiation.Most unwanted cells detached within 4 min of trypsin treatment.After 10 min, only encapsulated cells remained, achieving the selective removal of unnecessary cells.(2) Floating cells: Only targeted floating cells were exposed to light and immobilized on a coverslip.After photoencapsulation, PBS(-) washes removed unwanted cells.Trypsin treatment (6 min) dissolved the polymerized gel at 600 mJ/cm 2 , allowing for the collection of only the targeted cells.

Figure 1 .
Figure 1.Setup for encapsulating cells in GelMA hydrogels.Green and blue dots are respe target and unwanted cells.(a,b) Procedure for observing cells in a hydrogel after light irrad Blue region is a GelMA hydrogel.(c-g) Selection of target single attached cells encapsula GelMA by lysis with trypsin-EDTA.The principle of cell recovery is based on the susceptib trypsin.(h-l) Selection of target single suspended cells.The principle of cell selection is ba the susceptibility to flow.

Figure 1 .
Figure 1.Setup for encapsulating cells in GelMA hydrogels.Green and blue dots are respectively target and unwanted cells.(a,b) Procedure for observing cells in a hydrogel after light irradiation.Blue region is a GelMA hydrogel.(c-g) Selection of target single attached cells encapsulated in GelMA by lysis with trypsin-EDTA.The principle of cell recovery is based on the susceptibility to trypsin.(h-l) Selection of target single suspended cells.The principle of cell selection is based on the susceptibility to flow.
represents the negative control conditions without any treatment.Micro 2024, 4, FOR PEER REVIEW 5

Figure 6 Figure 4 .
Figure 4a,b demonstrates the biodegradation of GelMA hydrogels with and without encapsulated cells after 5 days.The gel containing cells exhibited significant degradation, while the cell-free gel remained largely intact.While the cell-free gel showed a slight decrease in the area, it was not statistically significant.Micro 2024, 4, FOR PEER REVIEW 6

Figure 4 .
Figure 4. GelMA degradation and proliferation.Proliferation of cells in 5%w/v GelMA hydrogel with a size of 100 µm × 100 µm.(a) Images of cells on Day 5.The arrows in the figure indicate that HeLa cells and cell division were observed.Three cells were encapsulated in the gel on Day 0. (b) Gel degradation without cells on Day 5. (c) Time series gel area with/without cells.A square pattern of 100 µm × 100 µm was used for this experiment.(d) Increase in cell number from Day 0 to Day 5.

Figure
Figure 5a,b, respectively, presents observations of HeLa cells encapsulated in the GelMA hydrogel over 2 days with initial cell number of 3 and 5.In Figure 5a, a cell in the upper left corner exemplifies cell division occurring between Day 0 and Day 1, while the lower right corner shows cell division from Day 1 to Day 2. The hydrogel appears slightly deformed 2 days after cell division, suggesting some biodegradation by the cells.

Figure 4 .
Figure 4. GelMA degradation and proliferation.Proliferation of cells in 5%w/v GelMA hyd with a size of 100 µm × 100 µm.(a) Images of cells on Day 5.The arrows in the figure indicat HeLa cells and cell division were observed.Three cells were encapsulated in the gel on Day Gel degradation without cells on Day 5. (c) Time series gel area with/without cells.A square pa of 100 µm × 100 µm was used for this experiment.(d) Increase in cell number from Day 0 to D

Figure 5 .
Figure 5. Time-lapse images of HeLa cells encapsulated in the GelMA hydrogel from Day 0 to 2. (a) Three cells and (b) five cells on Day 0, with single cells circled in black dashed lines.

Figure 5 .
Figure 5. Time-lapse images of HeLa cells encapsulated in the GelMA hydrogel from Day 0 to Day 2. (a) Three cells and (b) five cells on Day 0, with single cells circled in black dashed lines.

Figure
Figure 6a-d depicts a graph tracing the coordinates of the hydrogel corners and encapsulated cells, with the origin at a grid intersection.Cells remained confined within the gel, even when encountering the edge, and tended to move inward.Figure 6e shows the measured displacement of cells from their initial positions.The average cell migration was 12.2 µm ± 7.6 µm (N = 14) on Day 1 and 16.1 ± 9.6 µm (N = 14) on Day 2. A stiffer GelMA hydrogel might potentially reduce cell displacement, as reported in [36].

Figure 6 .
Figure 6.Motility analysis of HeLa cells encapsulated in GelMA hydrogel for 2 days.(a-d) Trajectories of cells with initial cell numbers ranging from 2 to 5. Gray areas indicate GelMA hydrogel.(e) Average cellular displacement on Day 1 and Day 2.

Figure 7 .
Figure 7. Collection of cells encapsulated in GelMA photocured at 800 mJ/cm 2 by trypsin treatment.(a) Immediately after irradiation.(b) Four min and (c) ten min after the addition of trypsin.The four cells remained trapped in the GelMA hydrogel.

Figure 6 .
Figure 6.Motility analysis of HeLa cells encapsulated in GelMA hydrogel for 2 days.(a-d) Trajectories of cells with initial cell numbers ranging from 2 to 5. Gray areas indicate GelMA hydrogel.(e) Average cellular displacement on Day 1 and Day 2.

Figure 6 .
Figure 6.Motility analysis of HeLa cells encapsulated in GelMA hydrogel for 2 days.(a-d) Trajectories of cells with initial cell numbers ranging from 2 to 5. Gray areas indicate GelMA hydrogel.(e) Average cellular displacement on Day 1 and Day 2.

Figure 7 .
Figure 7. Collection of cells encapsulated in GelMA photocured at 800 mJ/cm 2 by trypsin treatment.(a) Immediately after irradiation.(b) Four min and (c) ten min after the addition of trypsin.The four cells remained trapped in the GelMA hydrogel.

Figure 7 .
Figure 7. Collection of cells encapsulated in GelMA photocured at 800 mJ/cm 2 by trypsin treatment.(a) Immediately after irradiation.(b) Four min and (c) ten min after the addition of trypsin.The four cells remained trapped in the GelMA hydrogel.