Senescence chips for ultrahigh-throughput isolation and removal of senescent cells

Abstract

Cellular senescence plays an important role in organismal aging and age-related diseases. However, it is challenging to isolate low numbers of senescent cells from small volumes of biofluids for downstream analysis. Furthermore, there is no technology that could selectively remove senescent cells in a high-throughput manner. In this work, we developed a novel microfluidic chip platform, termed senescence chip, for ultrahigh-throughput isolation and removal of senescent cells. The core component of our senescence chip is a slanted and tunable 3D micropillar array with a variety of shutters in the vertical direction for rapid cell sieving, taking advantage of the characteristic cell size increase during cellular senescence. The 3D configuration achieves high throughput, high recovery rate, and device robustness with minimum clogging. We demonstrated proof-of-principle applications in isolation and enumeration of senescent mesenchymal stem cells (MSCs) from undiluted human whole blood, and senescent cells from mouse bone marrow after total body irradiation, with the single-cell resolution. After scale-up to a multilayer and multichannel structure, our senescence chip achieved ultrahigh-throughput removal of senescent cells from human whole blood with an efficiency of over 70% at a flow rate of 300 ml/hr. Sensitivity and specificity of our senescence chips could be augmented with implementation of multiscale size separation, and identification of background white blood cells using their cell surface markers such as CD45. With the advantages of high throughput, robustness, and simplicity, our senescence chips may find wide applications and contribute to diagnosis and therapeutic targeting of cellular senescence.

Keywords: anti-aging; cellular senescence; mesenchymal stem cells; microfluidic chip; size separation; total body irradiation.

Figure 1

Design and working mechanism of senescence chip. (a) Illustration of two types of chips: (i) a chip with a 3D filter array and a cell trap array for capture and single‐cell analysis of senescent cells in blood; and (ii) a chip with a 3D filter array connected with a tubing at the outlet for removal of senescent cells from blood. Zoom‐in regions show schematic separation and trapping of RBC, WBC, and senescent cells, along with two types of pillar shapes A and B. (b) Mechanism of size‐based cell separation with a 3D filter array: (i) the top view of the filters with force analysis on the x and y directions; (ii) the side view of the filters on the x and z directions; and (iii) the perspective view of the filters on the x, y, and z directions. (c) Images of the experimental setup and operation: (i) an actual‐size image of a senescence chip relative to a US dime; (ii) the experimental setup showing tubing connections and pumps; and (iii) a senescence chip in operation of processing whole blood samples. Scale bar represents 5 mm in (c‐iii). RBC: red blood cell; WBC: white blood cell

Figure 2

Operation of senescence chip. (a) Time‐lapse images showing: (i) a bead and (ii) a cell, roll down on a 3D filter array. (b) (i) Images showing undiluted whole blood passes through a 3D filter array without clogging; (ii) Stacking images showing complete separation of 18‐μm beads from 10‐μm beads (left), and separation of mesenchymal stem cells (MSCs) from undiluted whole blood (right). (c) Images of cell trap array located at: (i) blood outlet and (ii) senescent‐cell outlet, after separation of MSCs from whole blood. Cells with blue color are senescent cells (SA‐β‐gal positive); (iii) Phase‐contrast and fluorescence imaging of CD45 labeling for identification of senescent MSCs and WBCs. Scale bars represent 50 μm in (a) and (c), 150 μm in (b‐i), and 100 μm in (b‐ii), respectively. (a)‐(c) are shown as the corresponding zoom‐in regions on a schematic senescence chip for presentation clarity

Figure 3

Validation of senescence chip for size‐based separation. (a) Schematic of a senescence chip for characterization with beads or cells. (b) Recovery of beads from outlet (iii), for four sizes of beads (6 μm, 10 μm, 15 μm, and 18 μm) mixed to characterize three types of senescence chips (z‐direction only filter, 4‐μm 3D filter, and 13‐μm 3D filter) at three flow rates (1 ml/hr, 3 ml/hr, and 5 ml/hr). (c) Recovery of WBCs isolated from whole blood from outlet (iii), with three types of senescence chips at three flow rates as in (b). (d) Recovery of basal mesenchymal stem cells (MSCs) from undiluted whole blood at outlet (iv), with three types of senescence chips at a flow rate of 3 ml/hr. WBCs: white blood cells

Figure 4

Application of senescence chip for analysis of senescent cells in biofluids. (a) SA‐β‐gal staining of mesenchymal stem cells (MSCs) cultured on a 12‐well plate. The MSCs were treated with different doses of hydrogen peroxide (H₂O₂, 0, 100, 200 μm) and X‐ray (0, 1, 4 Gy), and analyzed 3 days and 6 days after the treatments. Cells stained blue are SA‐β‐gal positive. SA‐β‐gal: senescence‐associated beta‐galactosidase. (b) Quantitation of SA‐β‐gal staining of MSCs on culture dish (i) and MSCs isolated from human whole blood on the senescence chip (ii). The percentage of SA‐β‐gal positive was calculated for the blue‐stained MSCs among the total MSCs. (c) Isolation and analysis of senescent cells from mouse bone marrow after TBI of 0, 1 Gy, 4 Gy, and 6.5 Gy X‐ray radiation (n = 4), respectively. TBI: Total body irradiation. Scale bar represents 100 μm in (a)

Figure 5

Application of senescence chip for removal of senescent cells from whole blood. (a) Schematic of removal of senescent cells from whole blood, using a 13‐μm 3D filter. (b) Cell size distribution of basal mesenchymal stem cells (MSCs) and senescent MSCs, 3 and 6 days after treatment with different doses of hydrogen peroxide and X‐ray. (c) Enrichment of senescent cells at outlet (iv) using a 13‐μm 3D filter senescence chip. In comparison, original MSCs without separation, MSCs directly captured on a cell trap array without a 3D filter, and MSCs processed on a senescence chip with a 4‐μm 3D filter were also studied. (d) Removal of senescent cells from undiluted whole blood using a 13‐μm 3D filter senescence chip via outlet (iv). SA‐β‐gal: senescence‐associated beta‐galactosidase

Figure 6

Ultrahigh‐throughput senescence chip for removal of senescent cells from human whole blood. (a) Schematic of the high‐throughput senescence chip. Five large‐dimension channels are stacked and integrated for parallel processing. (b) Image of a high‐throughput senescence chip compared to a regular‐size single‐unit device. (c) Cross‐sectional view of the multilayer and multichannel senescence chip showing integration of five channels in five vertical layers. (d) Microscope images showing mesenchymal stem cells (MSCs) before separation, spiked in blood, and after separation. (e) Quantification of the numbers of basal MSCs and senescent MSCs spiked into whole blood, before and after removal of senescent MSCs using our senescence chip. Scale bars represent 10 mm in (b) and 50 μm in (d)