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A Lab-Made Wound Maker for Analysis of Cell Migration in a 96-Well Plate
Korean J Clin Lab Sci 2020;52:53-61  
Published on March 31, 2020
Copyright © 2020 Korean Society for Clinical Laboratory Science.

Tae Bok Lee1, Hwa Ryoung Kim2, Seo Young Park3

1Confocal Core Facility, Center for Medical Innovation, Seoul National University Hospital, Seoul, Korea
2Department of Biomedical Engineering, Seoul National University Hospital, Seoul, Korea
3Department of Research and Experiments, Center for Medical Innovation, Seoul National University Hospital, Seoul, Korea
Correspondence to: * Seo Young Park
Department of Research and Experiments, Center for Medical Innovation, Seoul National University Hospital, 71 Daehak-ro, Jongno-gu, Seoul 03082, Korea
E-mail: psy0305@snuh.org
* ORCID: https://orcid.org/0000-0003-3953-1653
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Cell migration is a central process for recovering from wounds triggered by physical distress besides embryogenesis and cancer metastasis. Wound healing assay is widely used as a fundamental research technique for investigation of two-dimensional cell migration in vitro. The most common approach for imitating physical wound in vitro is mechanical scratching on the surface of the confluent monolayer by using sharp materials. The iron metal pin with a suspension spring for fine adjustment of the orthogonal contact surface between the scratching point and the individual bottom of multi-well plate with planar curvatures were adopted for the creative invention of a 96-well plate wound maker. While classic tips drew diverse and zigzag scratching patterns on the confluent monolayer, our wound maker displayed synchronized linear wounds in the middle of each well of a 96-well plate that was seeded with several cell lines. Given that several types of multi-well plates commercially available are compatible with our lab-made wound maker for creating uniform scratches on the confluent monolayer for the collective cell migration in wound healing assay, it is certain that the application of this wound maker to the real-time wound healing assay in high content screening (HCS) is superior than utilization of typical polypropylene pipette tips.
Keywords : Cell migration, Recovering, Scratching, Synchronized, Wound healing
INTRODUCTION

The epithelial cells as a frontline defense mechanism cover the outer surfaces of skin, inner surface of internal organs and blood vessels throughout the body in order to protect the host in case various accidents occur caused by chemicals, heat, UV radiation, microorganisms and physical injuries. When this protective tissue is damaged, replacement by newly generated tissue called “wound healing” is quickly processed to restore the defense function [1]. The wound healing assay is a fundamental research technique for investigation of two-dimensional cell migration in vitro. Fibroblasts are crucially involved in the event of normal wound healing as a key modulator by breaking down the fibrin clots, fostering extracellular matrix (ECM) and building collagen structures which support surrounding cells for effective wound healing [2]. Cell migration is the common event for recovering from the wound triggered by physical distress and that has been analyzed for a score of years by scratching the cell layer with simple experimental tips varying from 10 to 100 μL in size depending on the severity of aimed wounds which mimics physical scratches. Nowadays, more often the cell migration analysis is experimentally applied not only to wound healing, mucosal repairing and epithelial-mesenchymal- transitions under normal development but also embryogenesis, neurogenesis, angiogenesis and in vitro cancer cell invasion [3]. Previously, phagokinetic track assay for single cell migration, and Boyden, Zigmond and Dunn chambers for chemoattractant migration assay have been widely used for in vitro studies of null cell migration without wounding recovery process [4, 5]. On the other hand, many studies are focusing on wound healing process wherein the consequential active movement of cell fronts towards empty space, which is scraped out of confluent monolayer by sharp materials imitating physical injuries, vividly observed [6]. Moreover, the living cells secrete defense molecules such as antimicrobial peptides (AMPs) and ECM proteins for anti-inflammation and fast recovery from wound [7, 8]. Given that quantifying analysis of cell migration induced by physical scratches duly imitates the actual wound healing process, a metallic wound maker with firm hardness for accurate linearity of scratches and sustainable tension spring for pressure balance given to each single well in the multi-well plate should be in consideration for experimental reproducibility of wound scratches and image data quantification compared to manual scratches with polypropylene pipette tips which demonstrate diverse, nonlinear and irregular wound patterns. In this article, we introduce a 96-channel mechanical wound maker mainly designed for CellCarrier-96 Ultra (PerkinElmer, Germany) microplate and also compatible to a variety of commercially available multi-well plates, and demonstrate its spontaneous performance with live cell imaging evaluation by high content screening (HCS) machine.

MATERIALS AND METHODS

1.Cell culture and High content screening (HCS) analysis

Fibroblast, HCC827, HEK293T, U8MG and HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Welgene, Korea) supplemented with 10% Fetal Bovine Serum (FBS, Gibco, USA) and 1× Penicillin- Streptomycin antibiotics (Welgene). For multi-well culture, cells were dispensed into a 96 well plate (CellCarrier-96 Ultra, PerkinElmer, Germany) or (Cat# 30096, SPL Life Sciences, Korea) according to the recommended seeding density (0.01×106). The plate was incubated at 37°C, 5% CO2 for two days consecutively. Wound healing ability was assessed by cell migration analysis of HCS (Operetta CLS, PerkinElmer) from the wounded edge to empty area. Cells were imaged using z-stack function to find the best focus area and each well was analyzed and showed with the multi-field stitching image in addition to bar graphs combined with merged realistic plate images. Cell count and tracking based on plate images were analyzed using digital phase contrast (DPC).

2.Layout and experimental design of lab-made wound maker

Pins with a diameter of 0.9 mm were produced by assembling the upper and lower part with suspension spring for fine adjustment between individual pin’s height and bottom surface of multi-well plate. The 96 pins were then planted into the transparent acrylic plate (125×90×16 mm) as per the dimensions of 96-well plate described at the insert sheet provided by commercially available multi-well plates. Guide rails were designed for smooth, straight and linear movement of wound maker along the both up and down sides of the plate. Plate holder was simply made of black acrylic plate for fixing the multi-well plate by attaching several bars surrounded tightly without interspace (Figure 1).

Fig. 1. Lab-made mechanical wound maker. (A) Lab-made mechanical wound maker composed of 96 spring pins planted on the transparent triple stacked acrylic plates and guide rail for prevention of scratching deviation while moving on the surface of a multi-well plate. (B) Close-up image of wound making pin. (C) Rearrangement of wound making pins in A, B, G, H rows and center integrated type for diverse application in wound healing assay. (D) An image of ready for making mechanical uniform wounds on a 96-well plate which is installed on the plate holder composed of opaque acrylic plate with four-sided panel. (E) An approximately calculated measures of 96-well plate layout for commercially available common plates and scratching spring pin. (F) A section plan describing perpendicular movement of iron metal pin with suspension spring along the well bottom of the plate.

3.Cell labeling for live imaging and making wounds

Fibroblast, HEK293T, U87MG and HeLa cells were seeded into a 96 well plate and after 48 hours incubation, cells were labeled with Calcein AM; cell- permeant dye (C1430, Thermo, USA) for 30 minutes. The plate and each well was rinsed twice with 1× Phosphate buffered saline (PBS) and added with pre-warmed cell culture media. In order to give consistent and homogenous wounds to each 96 wells, the plate was fixed on the plate holder and covered by the lab-made wound maker. By pressing the acrylic plate with moderate force carefully slid the wound maker along the guide rail and plate holder to the end of the well. Each wound was confirmed by naked eyes using widefield microscope (IX50, Olympus, Japan) beforehand and imaged using confocal laser scanning microscope (TCS SP8, Leica, Germany).

4.Sterilization of wound making pins

Wound making pins were pulled out of the transparent acrylic plate by tightly holding each pin with sterilized pliers and secured in the stainless steel cotton cylinder for autoclave at 121°C for 15 minutes. After which, pins were subjected to dehumidification under dry oven, overnight. In biosafety cabinet (BSC), each pin was planted onto the transparent acrylic plate using sterilized pliers. Acrylic plates were kept in BSC and regularly sanitized using 70% ethanol, UV irradiation and contamination prevent solution; Bioguard-S (#1010-1000, Biowest, MO, USA).

RESULTS

1.Comparison of wound modality scratched by lab-made wound maker and classic tips

Confluence of fibroblast cells were evenly 90∼100% in a 96-well plate in control. A wound maker simultaneously formed regular linear scratches in the middle of each well of the 96-well plate where fibroblast cells were fully seeded and labeled with green fluorescence. On the other hand, commercially available classic tips showed irregular and broad wounds left in the multi- well plate (Figure 2A). In the realistic view of multi-well plate under magnification, details of cells and patterns of scratches were analyzed. Compared to the wound maker, scratching patterns made by classic tips are diverse, complicated and somewhat wider than wound maker (Figure 2B). The scratched area of each single well was quantitatively analyzed and the results were represented as an open area. The open space created by wound maker was mostly monotonous compared to the diverse wounds scratched by classic tips. Similar results were also represented in cell area and confluence since open area, cell area and confluence are arithmetically all connected in the confined well in size. Nevertheless, a notable single deviation was observed in both cell area and confluence in the wounds made by classic tips (Figure 2C).

Fig. 2. A realistic plate view with magnified well images before and after making in vitro wounds. (A) Realistic overview images of confluent fibroblast monolayer labeled with calcein AM colored green on a 96-well plate before and after making in vitro mechanical wounds by lab-made wound maker or commercially purchased polypropylene pipette tips. (B) Magnification of selected well images. (C) Quantitative data analysis based on open area which represents scratched region of each row. The average measurements of cell area and confluence in the twelve consecutive wells after making wounds are quantitatively presented.

2.Superficial artifact on the surface of coated and non-coated plate

Fibroblast cells were seeded on the tissue-culture (TC) treated cyclic olefin copolymer (COC) bottom multi-well plate in order to investigate surficial scratching artifact consequently triggered by lab-made wound maker. As shown in Figure 3A, no significant differences were observed in the light of digital phase contrast (DPC) images both of classic tip (100 μL) and wound making pin. Interestingly, cell segments were more obvious on the scratched region of classic tip (100 μL) than wound making pin. On the other hand, bright field (BF) images of HCC 827 cells on the polystyrene multi-well plate showed clear scratches on the surface besides several wandering cells detached out of original settlement and there was no significant dissimilar between classic tip (100 μL) and wound making pin (Figure 3B).

Fig. 3. Comparison of scratching artifact on the surface of multi-well plate. (A) Seamless stitching tile scan images of digital phase contrast (DPC) of fibroblast on the tissue-culture (TC) treated cyclic olefin copolymer (COC) material after making physical wounds with classic tip (100 μL) and wound making pin. (B) Bright field (BF) images of HCC 827 cells after scratching the surface of the polystyrene multi-well plate with classic tip (100 μL) and wound making pin.

3.Live cell imaging of in-vitro wound healing

Regular and linear wounds created by the wound maker showed stable and all-around recovery from the parallel edges of wounds towards open area. On the other hand, irregular and ripped scratches by the polypropylene pipette tip demonstrated asymmetric and inconsistent recovery from the oblique and distorted edges of wounds towards open area with various patterns in the live cell imaging (Figure 4).

Fig. 4. Live cell imaging of in vitro wound healing process. Real-time fluorescent images captured by high content screening (HCS) machine (Operetta CLS, PerkinElmer) for 24 hours after making wounds on the confluent fibroblast monolayer. (A) Live cell images captured at 6 consecutive time points; 0, 4, 8, 12, 16 and 24 hours after giving mechanical wound with lab-made wound maker. (B) Live cell images of polypropylene pipette tip. Scale bar, 1 mm.

4.Precise and accurate mechanical wounds regardless of cells types compared to polypropylene pipette tips

To see whether a lab-made wound maker shows similar results; synchronized, linear and precise pattern of scratching wounds, on the different cell type with higher confluence, multi-well plates were filled with HeLa cells followed by manual scratches with commercially purchased polypropylene pipette tips or semi-automatic mechanical scratches using the lab- made wound maker. As shown in Figure 5A and B, each pipette tip used for 100 μL pipetting from different manufacturers demonstrates diverse scratching pattern and width of wounds compared to uniform wounds created by the lab-made wound maker. Wound linearity was calculated by measuring a degree of curvature of open area generated by mechanical scratching with lab-made wound maker or manual scratching with polypropylene pipette tips. Indeed, the lab-made wound maker presented synchronized horizontal linear scratches closed to “0” degree. On the other hand, manual scratches showed lopsided curved patterns with various bend angles much higher than the one presented by lab-made wound maker (Figure 5C). There was no significant difference in wound width among lab-made wound maker and several polypropylene pipette tips; however, the typical width created by each scratching material differs from 700∼800 mm depending on the thickness of endpoint of each material (Figure 5D). Different cell lines were adopted to see whether the lab-made wound maker demonstrates synchronized linear scratching results alike. As shown in Figure 5E, the lab-made wound maker produced uniform wounds on the diverse confluent monolayers formed by four different cell types: fibroblast, HEK293T, U87MG and HeLa, compared to the irregular and nonlinear wounds made by manual scratches.

Fig. 5. Diverse scratching modalities imparted by individual characteristics of polypropylene pipette tips and cell types. (A) Synchronized mechanical wounds imparted by lab-made wound maker on the multi-well plate with confluent monolayer of HeLa cells. (B) Uneven and irregular scratches made by each commercially available polypropylene pipette tip; Axygen TF-100, Gilson Pipetman E200 and Eppendorf 0030 073.428. (C) Quantification analysis of open area linearity based on scratching curvature and angle thereof. (D) Wound widths of four different conditions: wound maker, Axygen TF-100, Gilson Pipetman E200 and Eppendorf 0030 073.428. (E) Scratching modalities of lab-made wound maker and polypropylene pipette tip on the different cell lines: fibroblast, HEK293T, U8MG and HeLa cells. Data are expressed as the mean ±SD of sextuplicate determination. ***P < 0.001.
DISCUSSION

The predicaments caused by unsynchronized diverse wounds which are commonly observed in vitro wound scratch and healing experiments in a multi-well plate where all scratches are subjected to homogeneously linear pattern for unbiased and impartial assessment of real time wound recovery drew our attention to devising a simple lab-made wound maker. Different and various wounds in shape and size in one set of collective cell migration experiment for wound repair or cancer metastasis may lead to misconception of data analysis when it comes to the evaluation of wound recovery rate based on different given condition [9-11]. In our previous studies, making uniform in vitro wounds on the monolayer with same degree of cell confluence by scratching the surface using commercially available polypropylene pipette tips were hard in the aspect of accuracy and reproducibility [12, 13]. By adopting uniform pins commercially available made up of inner suspension spring and iron metal frame combined with a stainless steel endpoint, facilitated producing a 96-well plate wound maker by planting each pin along the column and row on the triple stacked acrylic plates as per the layout of multi-well plate (Figure 1). The classic tips made up of polypropylene, a hydrophobic polymer for ultra-low retention of liquid inside a wall, are mostly flexible and resilient in bending by steady pressure and that characteristic interrupts creating homogeneously synchronized wounds on the monolayer surface due to several factors: physical tremor, individual experience and distraction when consecutively scratching the restricted area in a confined round well less than 1 cm in diameter. On the other hand, our wound maker synchronously scratches monolayer and form uniform open area regardless of individual differences in experience and physical hindrance since all pins are in one plate and simultaneously move towards same direction under orthogonal even pressure (Figure 2). Purchasing commercially available finished wound scratching products costs researchers relatively high price compared to lab-made wound maker (Table 1). There was another novel approach on wound healing assay through the evaluation of cell migration into the gap created by silicon compartment attached onto the 12-well plate with confluent cell monolayer which is consequentially followed by the detachment of insert and that generates a clean cell-free space approximately 500 mm in width as a pseudo-wound without physical scratching [8, 14, 15]. Although the cell-free region called pseudo-wound created by physical barrier does not manifest the tissue injury followed by secretion of subordinate cellular component necessarily related to the wound healing processes, it seems more focused on the collective cell migration in cancer metastasis, inflammation and angiogenesis where the artificial cell injuries are not involved [16, 17]. Our wound maker is only designed for evaluation of collective cell migration in wound healing process by scratching the monolayer surface on a 96-well plate, simultaneously and synchronously. Furthermore, the individual suspension spring of scratching pins provides a perpendicular flexibility and advisable adoptability to the diverse bumpy thickness and irregular flatness of the individual well found in low-quality multi-well plates. Thus, several multi-well plates universally used for high content screening (HCS) are also acceptable for in vitro real-time wound healing experiment by applying our simplified 96-well wound maker.

Specification and price comparison between lab-made wound maker and commercially available finished products

Product Specification Price (KRW) Source
Lab-made Individual spring pins Uncoated detachable pins 0.9 mm wound Autoclave and 70% ethanol 100,000 SNUH BME
AccuWound 96 scratch tool TeflonTM Coating flexible pins Fixed type (not detachable) 1.2 mm wound 70% ethanol 9,000,000 ACEA Bio (https://www.aceabio.com/wp-content/uploads/ accuwound-20181109-v1.5-PRINT.pdf)
AFIX96FP6W Uncoated detachable pins Diverse selection of pins (0.23∼1.58 mm wounds) Autoclave and 70% ethanol 20,000,000 V&P Scientific (http://vp-sci.com/products/pin-tools/ wounding-pin-tools/wounding-fixtures/afix96fp6w.html)
IncuCyte WoundMaker Fixed type (not detachable) 0.7∼0.8 mm wound 70% ethanol WoundMaker alone is not purchasable Sartorius (https://www.essenbioscience.com/media/uploads/ files/8400-0012-D00_-_Migration_Invasion_User_Manual3_Protocols.pdf)

Acknowledgements

None

Conflict of interest
None
Author’s information (Position)
Lee TB1, M.T.; Kim HR2, M.E.T.; Park SY3, M.T.
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