Trypsin-Assisted Cell Depletion Method for Wound Healing Assay

Di Yin

, Shihmo Yang

, Hongbo Zhang

and Wenjun Zhang

1,3,*

School of Mechanical and Power Engineering, East China University of Science and Technology, Shanghai, China

Biomedical Science and Technology Research Center, School of Mechatronic Engineering and Automation,

Shanghai University, Shanghai, China

Division of Biomedical Engineering, University of Saskatchewan, Saskatoon, Canada

Keywords:

Wound Healing Assay, Cell Depletion, Cell Patterning, Trypsin.

Abstract:

Wound healing assay is a commonly used method in the laboratory to study cell migration ability. Among the

methods used to create cell-free zone, the widely used method, called cell depletion, will leave a certain

amount of injured cells in the migration regions, which will have an impact on the subsequent healing

experiments. To this end, we present a trypsin-assisted cell depletion method in wound healing assay to create

cell-free zone without dead cells. This method could rinse the dead cell after applying depleting process

without interfering in the attachment of the living cells. All the operation process is accomplished by

commonly used equipment and drugs in biological experiments. The effect of the enzyme is controlled by the

ambient temperature and processing time. The debris of dead cells are easily detached and removed to avoid

the impact on wound healing assay. This method is expected to combine with other 2D and 3D cell patterning

methods to form a more reliable cell processing technique.

1 INTRODUCTION

The investigation of wound healing assay would

provide more information about cell migration and

cell-cell interaction (HE, 2020) for biologist to study

cellular mechanisms (Grada, 2017), tumor formation

and metastasis (Teleanu, 2019), and inflammation

models (Biglari, 2019). The first step in wound

healing assay requires creating an artificial cell-free

zone which have been well developed by researchers.

And most of these methods can be categorized as cell

depletion that inevitably causes damage to the cells

(Monfared, 2021). Meanwhile, the injured cells may

remain on substrate, which seriously affect the wound

healing assay of the rest living cells. In this article, we

developed a trypsin-assisted method to remove these

injured cells by controlling the efficacy of trypsin by

temperature and time. After comparison, the most

effective processing parameters were obtained. With

experimental verification, this method will have

negligible side effect on the cells that are prepared for

the subsequent wound healing assay. This approach

not only addresses the inherent disadvantage of cell

depletion, but also makes the process of creating cell-

free zone regions more stable and reliable.

2 METHOD

The device with stamping function used to culture

cell is shown in Figure 1. The top layer of the chip is

the Polydimethylsiloxane (PDMS) with pillars

dimensions of 100 μm height and a 400μm diameter

fabricated by mold, which is made by

photolithography technology. After the PDMS is

made, the two ends of the PDMS are punched to

fabricate the outlet and inlet of the chip. The middle

layer is a spacer with a hollow cavity made of laser-

cut Acrylic(PMMA) board. And the bottom layer is

the culture dish substrate used for cell attachment.

After the oxygen plasma and ultraviolet treatment,

these three parts are aligned and bonded together by

double-sided adhesive tapes and use heavy objects to

press the device for an hour to obtain a fully sealed

channel with 1mm height and 1cm width. The

stainless steel needles are inserted at both ends of the

channel, and the liquid in the channel can be replaced

through the Teflon tube which are connected with

syringes. The PDMS layer can be pressed down so

that the pillars in the central region can touch the

bottom of the channel and realize the stamping

function.

Yin, D., Yang, S., Zhang, H. and Zhang, W.

Trypsin-Assisted Cell Depletion Method for Wound Healing Assay.

DOI: 10.5220/0012032800003633

In Proceedings of the 4th International Conference on Biotechnology and Biomedicine (ICBB 2022), pages 483-487

ISBN: 978-989-758-637-8

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

483

Figure 1: Schematic diagram of cell culturing chip with stamping function. Three layers of the chip are bonded and sealed by

two double-sided adhesive tapes. The two ends are connected by tubes to load the nutrients and trypsin that are necessary for

the experiment. And both ends of the tubs should be sealed with syringe to prevent leakage of liquid. The device will remain

in an incubator except for the trypsin experiment which requires the temperature of the chip to be adjusted.

Before the experiment, the device was filled with

liquid 75% ethanol and exposed to ultraviolet light

overnight. After disinfection, 5mL phosphate

buffer(PBS) was injected into the channel for

cleaning. And the PBS was then evacuated from the

channel by air, and the device was placed in a sterile

vessel to be vacuumed as much gas as possible from

the chip. After 30 minutes, the device was moved to

a sterile environment, and 75% alcohol was re-

injected to drain the air bubbles in the channel. After

that, the channel was washed again with PBS, and the

chip is ready for use. In the process of the experiment,

in addition to trypsin, other liquid injected into the

chip, must be put into the incubator in advance, so as

not to create bubbles in the chip.

The principle of the trypsin-assisted method is

shown in Figure 2 by appending weakened trypsin

digestion steps to the traditional cell depletion. In our

device, Cells were seeded into the channel as shown

in Figure 2(a). After incubation for 24 hours, the cells

reached 80% confluent without any liquid flow. The

DMEM in the device was replaced by PBS. Then, the

pillars were pressed down towards the substrate as

shown in Figure 2(c). After 5 minutes, the stamp was

reset to the original position. And the trypsin was

injected into the channel to replace the PBS and let

stand for several minutes as shown in Figure 2(e).

Last, DMEM was gently introduced into the channel

to rinse the dead cells and resupply the cells with the

nutrients that are necessary for the subsequent

experiment as shown in Figure 2(f).

ICBB 2022 - International Conference on Biotechnology and Biomedicine

484

Figure 2: The procedure of the trypsin-assisted method. The dark blue boundary represents the structure around the cell culture

chamber, the black arrow represents the flow direction of the different liquid, and the yellow arrow represents the direction

of the stamping movement. The stamping process can be performed manually or with other devices. It should be noted that

the pressure cannot be too large, otherwise the PDMS will touch the cells out of the target cell-free zone.

3 RESULTS AND DISCUSSIONS

The micrographs of the cells during the procedure

was shown in Figure 3. Before stamping, PDMS was

washed away with PBS. And the morphology of cells

is shown in Figure 3(a). After stamping, it can be

observed that the morphology of cells out of cell-free

zone was not significantly different from that of the

cells before stamping. Compared to the

aforementioned cells, the cells below the stamp were

heavily stressed, and most of them were dead and thus

detached the substrate. But there were still a few

injured cells attached to the substrate, which are

mainly concentrated in the center and the edge of the

cell-free zone, as shown in Figure 3(b). The regions

and quantities of these cells are not constant, which is

not reliable for studying injured cells. It also can

affect other living cells around. Therefore, it needs to

be processed immediately. Subsequently, trypsin was

slowly injected into the channel and remained at a

certain temperature and time. These injured cells

would fall off naturally, which were rinsed out of the

channel with re-injecting DMEM. And the remaining

cells form a well-defined cell-free zone as shown in

Figure 3(c).

Figure 3: bright-field micrographcs of the cell morphology before and after stamping(a)(b) and after being treated with

trypsin(c). The scale bar is 100 μm.

To access the effect of trypsin, which was affected

by the ambient temperature and the standing duration,

a series of experiments were carried out. Cell activity

and cell density at different temperatures are shown

Trypsin-Assisted Cell Depletion Method for Wound Healing Assay

485

in Figure 4(a). With the increase of temperature, the

effect of Trypsin became stronger and a large number

of dead cells fell off, leading to the increase of cell

viability. But at the same time, the cell density

decreased with the increase of temperature. The effect

of different standing duration on cells is shown in

Figure 4(b). With the increase of standing duration,

the shedding of dead cells also leads to the increase

of cell viability, while the cell density decreases

greatly.

Figure 4: The effect of the trypin in different conditions such like ambient temperature(a) and standing time(b). The cell

density is represented by bars and the cell viability is represented by dotted lines.

In order to evaluate the growth of cells treated

with or without trypsin, we compared our method

with the wound healing of cells in the cell exclusion

method within 24 hours. Here, the cells of the

experimental group were treated with trypsin at 20 ℃

for three minutes while the control group was treated

with DMEM for three minutes at the same

temperature. The healing area of cells was recorded

every 6 hours. As shown in Figure 5. In each time

point, the wound healing area was very close to each

ICBB 2022 - International Conference on Biotechnology and Biomedicine

486

other and all achieved more than 0.95 at 24 hours.

Compared with the control group, the effect of trypsin

treatment on cells was almost negligible. And the

experimental results further verify the feasibility of

our method.

Figure 5: Wound healing area of the cells under treated or non-treated conditions. The light colored columns indicate cells

that have not been treated with trypsin, while the dark colored columns indicate cells that have been treated with trypsin. The

wound healing area at certain time points was normalized to diagram cell migration.

CONCLUSION

The experimental results showed that by adjusting the

temperature and time of trypsin, targeted cell

clearance could be achieved and well-defined cell-

free zone could be obtained. It also proved that the

method not only removed the negative effects of the

dead cells, but also ensured that the remaining cells

treated with the enzyme were no different from the

untreated cells in subsequent wound healing

experiments. This method overcomes the inevitable

defects in the process of cell depletion and it will

expand the application prospects of more cell

patterning methods.

ACKNOWLEDGMENTS

We are thankful to Prof. Zhang and the staff of her

Laboratory at East China University of Science and

Technology for their support. We also extend special

thanks to the staff of the Biomedical Science and

Technology Research Center at Shanghai University.

REFERENCES

A. Grada, M. Otero-Vinas, F. Prieto-Castrillo, Z. Obagi, V.

Falanga.(2017) Research Techniques Made Simple:

Analysis of Collective Cell Migration Using the Wound

Healing Assay, J Invest Dermatol, 137: e11-e16.

G. Shabestani Monfared, P. Ertl, M. Rothbauer. (2021)

Microfluidic and Lab-on-a-Chip Systems for

Cutaneous Wound Healing Studies, Pharmaceutics, 13.

H.E. Deal, A.C. Brown, M.A. Daniele. (2020)

Microphysiological systems for the modeling of wound

healing and evaluation of pro-healing therapies, J Mater

Chem B, 8: 7062-7075.

R.I. Teleanu, C. Chircov, A.M. Grumezescu, D.M. Teleanu.

(2019) Tumor Angiogenesis and Anti-Angiogenic

Strategies for Cancer Treatment, J Clin Med, 9.

S. Biglari, T.Y.L. Le, R.P. Tan, S.G. Wise, A. Zambon, G.

Codolo, M. De Bernard, M. Warkiani, A. Schindeler, S.

Naficy, P. Valtchev, A. Khademhosseini, F. Dehghani.

(2019) Simulating Inflammation in a Wound

Microenvironment Using a Dermal Wound-on-a-Chip

Model, Adv Healthc Mater, 8 e1801307.

Trypsin-Assisted Cell Depletion Method for Wound Healing Assay

487