Lossless Immunocytochemistry using Photo-polymerized Hydrogel Thin-films

Immunocytochemistry (ICC), or immunofluorescence microscopy, is an essential biological technique for phenotyping cells in both research and diagnostic applications. Standard ICC methods often do not work well when the cell sample contains a small number of cells (<10,000) because of the significant cell loss that occurs during washing, staining, and centrifugation steps. Cell loss is particularly relevant when working with rare cells, such as circulating tumor cells, where such losses could significantly bias experimental outcomes. In order to eliminate cell loss in ICC protocols, we present a method to encapsulate the cell sample in a photo-polymerized hydrogel thin-film. The hydrogel thin-film is permeable to antibodies and other ICC reagents, thereby allowing the use of standard ICC protocols without modification. The cell sample is physically constrained by the hydrogel at the bottom surface of a standard (unmodified) imaging microtiter plate, thereby enabling the acquisition of high-quality micrographs regardless of the properties of the cell sample or staining reagents. Furthermore, while standard ICC requires several centrifugation steps during staining and washing, our hydrogel encapsulation method requires only a single centrifugation step. This property greatly reduces the time required to perform ICC protocols and is more compatible with robotic platforms. In this study, we show that standard ICC and Cytospin protocols are extremely lossy (>70% loss) when the sample contains less than 10,000 cells, while encapsulating the cells using a permeable hydrogel thin-film results in a lossless ICC process.

because of the significant cell loss that occurs during washing, staining, and centrifugation steps. 23 Cell loss is particularly relevant when working with rare cells, such as circulating tumor cells, 24 where such losses could significantly bias experimental outcomes. In order to eliminate cell loss 25 in ICC protocols, we present a method to encapsulate the cell sample in a photo-polymerized 26 hydrogel thin-film. The hydrogel thin-film is permeable to antibodies and other ICC reagents, 27 thereby allowing the use of standard ICC protocols without modification. The cell sample is 28 physically constrained by the hydrogel at the bottom surface of a standard (unmodified) imaging 29 microtiter plate, thereby enabling the acquisition of high-quality micrographs regardless of the 30 properties of the cell sample or staining reagents. Furthermore, while standard ICC requires several 31 centrifugation steps during staining and washing, our hydrogel encapsulation method requires only 32 a single centrifugation step. This property greatly reduces the time required to perform ICC 33 protocols and is more compatible with robotic platforms. In this study, we show that standard ICC 34 and Cytospin protocols are extremely lossy (>70% loss) when the sample contains less than 10,000 35 cells, while encapsulating the cells using a permeable hydrogel thin-film results in a lossless ICC 36 process. 37 38 39 Introduction 41 Immunocytochemistry (ICC), or immunofluorescence microscopy, is an essential biological assay 42 that uses fluorescence-conjugated antibodies to label cells in order to phenotype them based on 43 protein expression and localization. This assay involves repeated exchange of reagents for cell 44 fixation, permeabilization, blocking, immunostaining, as well as additional buffer washes between 45 each step. When the specimen contains a large number of cells (typically >10 5 cells per ml), there 46 is sufficient cell density to form a pellet during centrifugation, which enables supernatant removal 47 by pipetting or decanting the fluid. However, when there are fewer cells, the cell density is too low 48 to pellet and many cells may be lost during each supernatant removal step. This issue is particularly 49 important when working with precious samples, where the specimen is limited, or where target 50 cells are rare. For example, detecting circulating tumor cells (CTCs) in the blood of cancer 51 patients [1][2][3][4] or fetal cells in maternal blood 5 , require immunostaining of exceedingly rare cells, 52 where the loss of potential target cells cannot be tolerated. 53 Numerous modifications of the conventional ICC protocol have been developed to prevent cell 54 loss. One approach is to chemically attach cells on a glass slide coated with an adhesive, such as 55 poly-L-lysine, fibronectin, or Cell-tak 6-8 , and then perform the ICC protocol directly on the glass 56 slide. This approach works well for adherent cells grown in culture, but the adhesives are typically 57 ineffective for primary cells or cultured suspension cells. An alternative approach is Cytospin™, 58 which physically adheres cells to a glass slide using centrifugal force 9,10 . While both primary cells 59 and cells grown in culture can be adhered to a glass slide, this process contributes to significant 60 cell loss. Specifically, when the cell number is relatively small (<10 5 cells per ml), previous studies 61 have reported losses of >75% 11 . Additionally, Cytospin is a serial process performed one sample 62 at a time, which significantly limits experimental throughput in screening studies 12 . Finally, while Cytospin deposits cells in a confined region on a slide, the deposition area typically requires 64 capturing many microscopy fields to image, which adds to the time required for imaging. 65 Therefore, when the sample contains a small number of cells, concentrating cells in a smaller 66 imaging area can significantly reduce imaging time. 67 Here, we present a method to prevent cell loss during ICC by encapsulating cells in a hydrogel 68 thin-film. This approach has been used previously by encapsulating cells in low-melt agarose 13 , 69 which forms a hydrogel matrix that is optically transparent and permeable to ICC reagents. 70 However, this approach has not been widely adopted because the viscosity of agarose solutions 71 which prevent the alignment of cells to a precision surface for imaging. Instead, the agarose 72 hydrogel must be sectioned to image the cells from each optical plane. In this study, we present a 73 cell encapsulation material that has lower density than typical cells in order to enable the alignment 74 of cells by centrifugation to a single layer on the bottom surface of a standard and unmodified 75 imaging microtiter plate. This material is permeable to immunoglobulins, optically transparent 76 with minimal coloration and auto-fluorescence, and mechanically robust to withstand repeated 77 washing. Through additional experiments, we show that the lossless ICC process is able to (i) 78 retain and stain 100% of the cell sample, (ii) confine the cell sample into a small area for rapid 79 high-quality imaging, and (iii) can be performed with only a single centrifugation step. 80

82
Our general approach is to mix cells in a pre-polymer solution that can be crosslinked into a 83 hydrogel upon ultraviolet(UV) light exposure. To enable lossless ICC, the prepolymer solution 84 must be less dense than cells to allow them to sink to the imaging surface via centrifugation. The 85 hydrogel thin film must have sufficient mechanical strength to withstand normal pipetting. Finally, 86 the hydrogel must be sufficiently thin and porous to allow the diffusion of immunoglobulins in a 87 reasonable timeframe (~1 hour)( Figure 1). 88

Density Testing 89
In order to align cells by centrifugation to a single layer on the bottom glass surface of the imaging 90 microtiter plate, the cell capture solution density must be less than that of typical cells. Given that 91 the lowest density cells are likely to be monocytes, which have a density between 1.067 and 1.077 92 g/ml 28 , the cell capture solution should be less than 1.067 g/ml in density. Also, the density must 93 be higher than 1 g/ml to sink and encapsulate the cells in bottom plane. We aimed the density of 94 cell capture solution as 1.058 g/ml. 95

Mechanical Strength Testing 96
The mechanical strength of the hydrogel thin-film is important for retaining its structural integrity 97 during pipetting. This property was tested by repeatedly pipetting 40 µl of PBS onto the surface of 98 the photopolymerized hydrogel until signs of structure disintegration, such as cracks, tears, and 99 delamination, began to be observable. The polymerized hydrogel thin-film had sufficient 100 mechanical strength to survive >100 rounds of repeated pipetting. 101

Porosity Testing 103
Immunoglobulins have an estimated size of ~14 nm 14,15 . The porosity of hydrogel thin-film must 104 be sufficiently large to permit diffusion of immunoglobulins to the cell sample in a reasonable 105 amount of time. Conventional method for producing macroporous hydrogels include freeze-106 drying, solvent casting, and gas forming 16-21 . While these methods have been used in tissue 107 engineering applications to produce hydrogels with >100 µm pores 22-26 , these hydrogels have poor 108 mechanical strength and image quality 27 , which makes them incompatible with immunostaining 109 of embedded cells. We evaluated whether ICC could be performed on cells embedded in lossless 110 hydrogel by embedding 22RV1 cancer cells and then using fluorophore-conjugated antibodies to 111 stain the extracellular EpCAM protein, and the intracellular cytokeratin proteins. The 90% of cells 112 were stained after 1 hour incubation. 113

Thickness Testing 114
In addition to porosity, the thickness of the hydrogel thin-film is important for determining the 115 time required for immunoglobulin diffusion. Immunoglobulin stains are introduced on the top 116 surface of the hydrogel and must diffuse to cells located at the bottom surface of the hydrogel, 117 which interfaces with the glass substrate. The thickness of the hydrogel thin-film can be controlled 118 by the UV light intensity and exposure time. Based on the Beer-Lambert law, UV light intensity 119 diminishes exponentially as it penetrates absorbing material. Therefore, UV light applied at the 120 bottom of the imaging plate polymerizes a hydrogel thin-film with thickness directly controlled by 121 exposure time. We tested hydrogel thin-film formation by exposure using a long-wavelength UV 122 LED (λ=375 nm) for 1, 3, 5, 7, or 10 seconds. We also tested hydrogel thin-film formation by 123 exposure using standard UV gel imaging system (λ=302 nm) for 5, 10, 15, 20, and 30 seconds.
The hydrogel thickness was then estimated by first focusing on a cell along the imaging plate 125 surface and then the top hydrogel surface. The z-position of each focal point was obtained from 126 Nikon NIS-BR software and used to estimate the distance between the two points. Three 127 measurements were performed for each experimental condition. Using an UV LED, exposure for 128 1 and 3 s failed to form a hydrogel, while 5, 7, and 10 s exposures produced hydrogels with 129 thickness of 100 +/-20 µm, 500 +/-20 µm, and 1000 +/-20 µm, respectively. Therefore, we 130 selected the 5 s as the optimal exposure time for UV LED source because it produced a stable 131 hydrogel while minimizing UV exposure and minimizing hydrogel thickness. When using the UV 132 gel imaging system, exposure <20 seconds failed to form a hydrogel, while 20 seconds exposure 133 produced 100 +/-20 µm thickness hydrogel and 30 seconds exposure produced 300 +/-20 µm 134 thickness hydrogel. Therefore, for gel imaging system, we used an exposure time of 20 seconds to 135 minimize hydrogel thickness. 136

Immunocytochemistry of Hydrogel Encapsulated Cell Samples 137
To evaluate this cell fixation method for use in immunocytochemistry, we first mixed the cell 138 sample with the prepolymer solution in a glass-bottom imaging well plate and then centrifuged the 139 well plate to align the cells on the surface of the glass. Next, the hydrogel thin-film is formed using 140 a 5 s UV exposure, at which point, the specimen is immunostained using standard ICC reagents 141

Cell Loss Comparison 159
We investigate cell loss during ICC resulting from convention protocol, Cytospin, and hydrogel 160 encapsulation. Using 22RV1 prostate cancer cells as a model, we generated a 10-fold dilution 161 series containing 10 to 10,000 cells, and then immunostained the samples using standard ICC, 162 Cytospin, and hydrogel encapsulation. To measure cell loss, we performed triplicate experiments 163 where cells from each specimen were enumerated by two independent reviewers before and after 164 ICC ( Figure 6). Cells stained using traditional ICC and Cytospin retained less than 50% of the 165 cells during immunostaining regardless of the number of starting cells in the sample. In contrast, 166 the hydrogel encapsulated cell sample retained 97-99% of input cells following immunostaining. 167 The small deviation from ideal likely resulted from incomplete staining rather than cell loss since 168 there are invariably a small fraction of a cell sample that will not stain. The most significant 169 difference in cell loss was observed when fewer than 100 input cells were stained. Under these situations, almost all (>84%) cells were lost using standard ICC and Cytospin. In fact, Cytospin 171 failed to retain any cells when there were only 10 cells in the initial sample. Together, these results 172 show that hydrogel encapsulation permits virtually lossless immunostaining that is robust 173 regardless of the starting number of cells in the sample. 174

175
This technical note presents a porous hydrogel thin-film for encapsulating cells during 176 immunocytochemistry in order to eliminate cell loss resulting from washing and centrifugation. 177 We show that this hydrogel thin-film is permeable to immunoglobulins, stable enough to withstand 178 pipetting, and allows immunostaining to be performed directly on standard imaging well plates. 179 Compared to standard ICC and Cytospin methods that become highly lossy for small cell samples 180 (<10,000 cells), this process is lossless and can be used to stain <10 cells in a well. Furthermore, 181 this process requires only a single centrifugation step, compared to >8 steps for standard ICC, 182 which greatly improves compatibility with robotic systems. Ultimately, this simple and novel 183 application of hydrogels for ICC could greatly improve small cell sample biological assays, such 184 as drug screening on primary cells and identification of rare cells, such as circulating tumor cells. 185

Chemicals and Hydrogel Preparation 188
The cell capture imaging reagent (LMR001) was purchased from MilliporeSigma. The

Cell Encapsulation 198
To encapsulate the cells in hydrogel, each cell suspension and 40 µL of PBS buffer was first loaded 199 into one well in a 384-high contrast imaging well-plate (Corning). Next, 6.5 µL of the cell capture 200 imaging reagent solution is pipetted gently with minimal mixing. The imaging well-plate was then 201 centrifuged for 3 minutes at 3800 rpm (Accuspin 1R, Fisher scientific), and immediately proceeded 202 to next step. 203

Photo-polymerization 204
To form a hydrogel thin-film, the previously prepared plate was exposed to UV light using a 375 205 nm UV LED (M375L3, Thorlabs) powered by a LED driver (LEDD1B, Thorlabs), or a cold cathod 206 fluorescent lamp (CCFL) UV lamp (λ=302 nm) in a gel imaging system (Gel Doc XR+, Bio-Rad). 207 For UV LED system, the center of the LED was aligned with the center of the each well with 0.5 208 mm gap and exposed for 5 seconds under drive current of 700 mA, which provides 470 mW output 209 power. For the gel imaging system, the location of CCFL UV lamp was pre-marked, and 3 rows 210 of well-plate were aligned on the center of each UV lamp. The exposure was controlled by Image 211 Lab software (Bio-Rad) same as regular DNA gel imaging with a 20 second exposure. 212

Cytospin Preparation 213
Cytospin was performed by depositing a 40 µL cell suspension directly onto a BSA-coated glass 214 slide using a cytocentrifuge (Cytospin 2, Shadon) at 700 rpm for 3 minutes with low acceleration.  immunostaining is permeable to immunoglobulins. 323