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Primary and Stem Cell Microarrays: Application as Miniaturized Biotesting Systems

  • Rebecca Jonczyk
  • Thomas Scheper
  • Frank Stahl
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1771)

Abstract

The deposition of living cells on microarray surfaces can be used to create physiologically relevant architecture in vitro. Such living cell microarrays enable the reconstruction of biological processes outside the body in a miniaturized format and have many advantages over traditional cell culture. The present protocol offers an option for the preparation and analysis of living primary and stem cell-based microarrays utilizing the standard microarray equipment (contact-free piezoelectric nanoprinter, microarray scanner), as well as microscopy. To produce living cell microarrays, we applied two kinds of mesenchymal stem cells (MSCs) isolated from umbilical cord and adipose tissue, as well as human umbilical vein endothelial cells (HUVECs) as model cells. We used live imaging microscopy for the online monitoring of cell spots in total size, staining of viable cells with Calcein acetoxymethyl ester (Calcein-AM) and treatment of MSCs with differentiation media to analyze the proliferation, viability, and differentiation potential of printed cells. This way, the general applicability of the established living cell-based microarray production was demonstrated.

Key words

Microarray technology Piezoelectric nanoprinting Living cell microarrays Primary human cells Mesenchymal stem cells Endothelial cells Online monitoring microscopy 

1 Introduction

In the 1990s microarrays became the standard tool for gene expression analysis. Combining high-throughput screening and miniaturization, this platform was applied to a variety of biomolecules and biological samples. Fluorescence/bioluminescence or label-free electrochemical signals are used to observe nondestructively recognition events on all microarray formats [1, 2, 3]. Since Ziauddin and Sabatini established the first living cell microarrays in 2001 [4], several research groups have developed further microarray systems of living mammalian cells [5, 6, 7]. Living cell-based microarrays are utilized to study functional and vital cellular responses while external stimuli activate the cells. This leads to intracellular signaling, the induction of transcription or translation of genes, cell proliferation or cell death, as well as differentiation. To ensure meaningful results, the microarray system itself—surface as well as preparation procedure—should not affect cellular physiology [5, 8]. Living cell microarrays are prepared in two different ways: either cell suspension is arranged to defined locations on the microarray, e.g., by printing [9], or microarray surface is modified in a way allowing cells to attach only at defined spots [10, 11]. Only in few studies a contact-free inkjet printer is utilized to position droplets of few picoliters of cell suspension at defined locations on a microarray [12, 13, 14] while high cell viability has been observed. We recently established a simple method to produce and analyze living cell-based microarrays with standard microarray equipment, which involves nanoprinter and fluorescence laser microarray scanner [15]. With the help of a piezoelectric noncontact nanoprinter we were able to transfer very small amounts of living model cells (1200 cells of A-549 lung cancer cell line and fewer) with high repeatability (spot-to-spot variation: ±8.6 cells) to modified microscope slides. The printed cells were cultivated, monitored, and analyzed with several methods ranging from online monitoring (incubator microscope) to cell viability assays, such as CellTiter-Blue® Assay, and to conventional cell staining assays, like DAPI (cell nuclei staining) or Calcein-AM (viable cell staining). The cells grew in clearly defined circular spots with a growth rate comparable to standard conditions [15].

Due to the low number of cells required per spot, this technique enables the use of limited test material, such as primary and stem cell samples, that are processed and analyzed using a variety of methods. In addition, printing of cells onto slides with coatings or topographic structures, as well as into microtiter plates, is easily feasible, resulting in microarrays, which provide an in vivo-like microenvironment. For example, such in vivo-like microenvironments enable drug discovery and development using human cells and help to reduce animal testing.

2 Materials

Prepare all solutions at room temperature using ultrapure water and analytical or cell culture grade reagents.

2.1 Cell Culture Experiments

  1. 1.

    Cryopreserved human mesenchymal stem cells (from umbilical cord: ucMSCs, adipose tissue derived: adMSCs): isolated earlier and characterized according to the minimal criteria established by the International Society for Cellular Therapy [16] (ISCT, adherence to plastic under standard cultivation conditions, differentiation into osteoblasts, chondrocytes, and adipocytes) [17, 18].

     
  2. 2.

    Cryopreserved human umbilical vein endothelial cells (HUVEC), which are pooled from multiple isolates.

     
  3. 3.

    Alpha Minimum Essential Medium (α-MEM, (−) nucleosides): For basal medium add 10.08 g α-MEM powder and 2.2 g sodium hydrogen carbonate to a 1 l graduated flask with about 800 ml distilled water (dH2O). Mix and make up to 1 l with dH2O and filter through a 0.2 μm pressure filtration unit (see Note 1 ). Store at 4 °C.

     
  4. 4.

    α-MEM culture medium: Mix α-MEM basal medium, 10% human serum, 0.5% gentamycin under sterile conditions. Store at 4 °C.

     
  5. 5.

    Endothelial Cell Growth Medium 2 (EGM2): Mix 97 ml EGM2, ready-to-use sterile liquid basal medium, 2.5 ml SupplementMix, 0.5 ml gentamycin under sterile conditions. Store at 4 °C.

     
  6. 6.

    Phosphate buffered saline (PBS): Dissolve 2 PBS tablets in 1 l dH2O and autoclave the solution. Store at 4 °C.

     
  7. 7.

    Sterile Disposables: Pipettes and pipette tips, T75 cell culture flasks, pasteur pipettes, 1.5 ml, 2.0 ml, 15 ml, and 50 ml tubes.

     
  8. 8.

    Determination of cell number and cell viability: Hemacytometer, Trypan Blue (0.2% in PBS).

     

2.2 Piezoelectric Noncontact Printing

  1. 1.

    Contact free piezoelectric nanoprinter (Gesim Nanoplotter NP2.1, Grosserkmannsdorf) for printing of cells equipped with a piezoelectric pipetting tip Nano-TipA J (Fig. 1).

     
  2. 2.

    Donor template: a sterilized PCR plate covered with sterile cover sheeting for multiwell plates.

     
  3. 3.

    Target for printing: coated glass objective slides, which were fixed in an incubation chamber of 96-well plate dimensions, designed in the workshop of the Institute of Technical Chemistry, Leibniz Universität Hannover (Fig. 1). This incubation chamber consists of three inner parts with two columns of eight holes each. It is closed with a cover for 96-well plates.

     
Fig. 1

Scheme of the preparation of primary and stem cell microarrays and cell monitoring. Cells were harvested, printed, and cultivated for a couple of days while they were monitored by the incubator microscope (1). Afterwards, cell viability using Calcein-AM staining (2) and an impact to the cells were analyzed (examination of their differentiation potential, 3)

2.3 Online Monitoring of Printed Cells via Incubator Microscope

  1. 1.

    Incubator microscope for online monitoring of printed cell spots in total, equipped with fourfold phase contrast objective and 3.8 MP CMOS sensor.

     
  2. 2.

    IrfanView is used to rename and convert the images, ImageJ is used to correct background signals and with VirtualDub software time-lapse videos are produced.

     

2.4 Endpoint Testing of Cell Viability

  1. 1.

    Calcein-AM working solution: Prepare stock solution of 1 mM under sterile conditions by resolubilizing 1 mg powder in 1 ml anhydrous dimethyl sulfoxide, aliquot the stock solution and store at −20 °C. Dilute the stock solution 1:30 in PBS (add 1 μl stock solution to 29 μl PBS) and additionally 1:10 in basal medium (add 270 μl basal medium to the first dilution) (see Note 2 ).

     
  2. 2.

    An inverse microscope equipped with a fluorescence source and a camera (see Note 3 ).

     
  3. 3.

    A fluorescence laser microarray scanner for possible detection and quantification applications with two lasers (Excitation 532 nm and 635 nm) (see Note 4 ).

     

2.5 Analysis of the Impact of Printing to MSCs by Examining Their Differentiation Potential

  1. 1.

    Osteogenic, chondrogenic, and adipogenic differentiation medium: ready-to-use media, 0.5% gentamycin (see Note 5 ).

     
  2. 2.

    Osteogenic differentiation (Alizarin Red solution): Dissolve 1.0% Alizarin Red S in 2.0% ethanol, use a 0.2 μm filter unit (see Note 6 ).

     
  3. 3.

    Chondrogenic differentiation (Alcian Blue 8GX solution): Dissolve 1.0% Alcian Blue 8GX in 3.0% acetic acid; subsequently use a 0.2 μm filter unit.

     
  4. 4.

    Adipogenic differentiation (BODIPY 493/503; 4,4-difluoro-1,3,5,7, 8-pentamethyl-4-bora-3a,4a-diaza-s-indacene): Prepare stock solution of 10 mM by resolubilizing 10 mg powder in 3.815 ml anhydrous dimethyl sulfoxide, aliquot the stock solution and store at −20 °C. Dilute the stock solution 1:2000 in PBS (add 1 μl stock solution to 1999 μl PBS) to prepare a 5.0 μM working solution.

     
  5. 5.

    DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) buffer: 12.11 g/l Tris-HCl pH 7, 8.766 g/l NaCl, 0.111 g/l CaCl2 (anhydrous), 0.101 g/l MgCL2 7*H2O, 0.1% Nonidet-P40 substitute, use a 0.2 μm filter and store at 4 °C.

     
  6. 6.

    DAPI staining solution: Prepare stock solution of 500 μg/ml by resolubilizing 1 mg powder in 2 ml sterile dH2O, store at 4 °C in the dark. Dilute the stock solution 1:500 in DAPI buffer (add 2 μl stock solution to 1998 μl dH2O) to prepare a 1.0 μg/ml working solution.

     

3 Methods

Carry out all procedures at room temperature and under sterile conditions unless otherwise specified. Perform all cultivation and incubation steps at 37 °C in a humidified environment with 5% CO2 unless otherwise stated.

3.1 Cell Culture Experiments

  1. 1.

    Grow MSCs in T75 cell culture flasks in 10 ml α-MEM culture medium and use them preferably for experiments in passages 2–12 (see Note 7 ).

     
  2. 2.

    Cultivate HUVECs in EGM2 and print them in passages 2–5 (see Note 8 ).

     
  3. 3.

    Reaching 80% of confluence, wash the cells in cell culture flasks with 10 ml PBS, which was warmed at 37 °C in water bath, and detach them by treatment with 2 ml Accutase® for 5 min.

     
  4. 4.

    Stop Accutase® reaction by addition of 3 ml warmed cell culture medium. Remove Accutase® by transferring cell suspension to a 15 ml tube and centrifugation for 5 min at 300 × g.

     
  5. 5.

    Resuspend cell pellet in 1–3 ml fresh medium (depending on pellet size) and count the cell number using a hemacytometer and Trypan Blue (20 μl cell suspension, 20 μl dye) to distinguish between viable (unstained) and dead cells (blue). For subcultivation transfer 2000–3000 MSC/cm2 or 5000–6000 HUVEC/cm2 to a new cultivation flask with fresh, warmed cell cultivation medium every third to fourth day. A cell suspension of 250,000 cells in 50 μl is used for printing while the amount of printed cells is regulated by the number of printed droplets (see Note 9 ).

     

3.2 Piezoelectric Noncontact Printing

  1. 1.

    Sterilize parts of the incubation chamber that get in contact with cells or medium by autoclaving. The parts of the outer chamber, as well as the cover for 96-well plates to close the sterile chamber are sterilized by incubation in 70% 2-propanol for 30 min.

     
  2. 2.

    Furthermore, 1 day before printing fix uncoated sterilized glass slides in an autoclaved incubation chamber. Dilute collagen solution (1 mg/ml Type I, aseptically processed) tenfold with sterile dH2O and coat each well of the microarray with 10 μg/cm2 collagen overnight at 4 °C. The next day, aspirate the solution and allow the surface to dry for 1–2 h (see Note 10 ).

     
  3. 3.

    Moisture the coated well surfaces of the microarray by addition of 50 μl culture medium per well directly before printing of cells (see Note 11 ).

     
  4. 4.

    Prepare a working plate, defining the target group attributes, such as the outer dimensions as well as the dimensions of the printing area itself (see Notes 12 and 13 ).

     
  5. 5.

    In addition, prepare a transfer list. Each well of the donor template, into which the cell suspension is transferred, is given an own target position within the printing area. The exact position of the pipetting tip within the target position is also defined (see Note 14 ).

     
  6. 6.

    Directly before the printing process, clean the nanoprinter with 70% 2-propanol, the nanoprinter is initialized and the pipetting tip is washed extensively with sterile dH2O (see Note 15 ).

     
  7. 7.

    Transfer cell suspension (50–100 μl) to the well of the donor template that was defined in the transfer list (see Note 16 ).

     
  8. 8.

    Perform the printing process using these settings: pulse width = 50 μs, frequency = 100 Hz, voltage = 75 V, delay = 1.00. The pipetting tip shall be dried before sample uptake, the formation of droplets shall be checked with stroboscope (camera) before the printing, 1.5 μl extra volume shall be used and an extra wash for 15 s shall be performed, the aspiration flow is set to 1 μl/s. The distance between the pipetting tip and the target is determined to be in maximum 7 mm at the spotting position and 15 mm during the move (see Note 17 ).

     
  9. 9.

    Place the prepared microarray with printed cells in the incubator for 1 h cautiously, watching out for preventing any agitation. Thereafter, add 150 μl fresh culture medium to the wells and cultivate the cells for several days and monitor them meanwhile. After the desired cultivation duration, perform biotesting and analytics of your choice.

     

3.3 Online Monitoring of Printed Cells via Incubator Microscope

  1. 1.

    Proliferation behavior of printed MSCs and HUVECs on coated microarrays is observable by capturing an image of the total cell spot utilizing a microscope in brightfield mode equipped with a fourfold phase contrast objective and placed in the incubator. This way, the cause of endpoint analysis results can be illuminated in more detail (Fig. 2).

     
  2. 2.

    Settings: White LED, 8–12%; gain, 9–17%; LED presnap on time 5.00 s (see Note 18 ).

     
  3. 3.

    The time interval between capturing image depends on the duration of cultivation. Here, every 2–5 min images are captured over a cultivation period of 2–3 days. Turn of LED automatically between capture intervals to prevent phototoxicity or photobleaching.

     
  4. 4.

    To correct the images in a batch and create a time-lapse video using VirtualDub it is necessary that all images are serially numbered and converted to JPEG format. Microscope images are often saved as TIF files, which are lossless in terms of quality. Therefore, rename and convert the single images of one folder using IrfanView for example, if necessary.

     
  5. 5.

    If necessary, correct all images with ImageJ by the pseudo-flat-field correction (blurring radius: e.g., 25) and by the unsharp-mask (σ: 2) to reduce background (see Note 19 ).

     
  6. 6.

    Using VirtualDub software time-lapse videos of the pictures are produced. By Drag‘n’Drop of the first image of one folder to the VirtualDub window, the software imports all serially numbered images of the folder automatically. Set a frame rate of 24 images per sec using the function “video” and “frame rate.” Use the compression “VirtualDub Hack” (x264 Codec). Afterwards, start the encoding process, and prepare and save the video.

     
Fig. 2

Proliferation of 1200 printed adMSCs (a, b) and 2000 printed HUVECs (d, e) on a living cell microarray. Images are captured by the incubator microscope equipped with fourfold phase contrast objective. a, b: LED 9%, gain 10%, interval 4 min, cultivation duration 3 days. d, e: LED 12%, gain 17%, interval 2 min, cultivation duration 2 days. a: 2 h after printing, b: 72 h after printing, d: 2 h after printing, e: 24 h after printing, c, f: QR-Codes for full videos

3.4 Endpoint Testing of Cell Viability

  1. 1.

    One or 2 days after printing the primary cells, biotesting can be performed. Here, the viability of printed cells is investigated using a live cell staining with Calcein-AM . In this way, all cells of the spot can be examined easily. Remove culture medium out of the wells carefully, add 100 μl of Calcein-AM working solution and incubate the microarray for 30 min.

     
  2. 2.

    Perform the imaging of stained cells via the inverse microscope directly in the microarray incubation chamber system using the fluorescence source and a filter set for fluorescein isothiocyanate (FITC), e.g., BP470-490 (exciter filter), BA515 (barrier filter).

     
  3. 3.

    Carry out the detection of stained cells using a fluorescence microarray scanner after removing the incubation chamber. It was shown elsewhere that a mounting of the microarray with a protection buffer and a coverslip, which is sealed using construction adhesive, results in an increase of fluorescence signal quality [19]. To show the proof-of-principle here, PBS is used.

     
  4. 4.

    Place the microarray upside-down into the reading chamber and use these settings for all scanning processes: Pixel size = 5 μm, lines to average = 1, focus position = 75 μm and scan power = 33%.

     
  5. 5.

    Initially, perform a preliminary scan to determine the scan area and the photomultiplier (PMT) gain. The PMT gain is varied between 350 and 550, resulting in images of high fluorescence without any saturation of signals (Fig. 3).

     
  6. 6.

    Images are saved in grey scale. Therefore, false color (green) is added to the scan images using the LUT mode in ImageJ to get an RGB image of the scans.

     
Fig. 3

Scans of viable, Calcein-AM stained primary cells (a: HUVECs, c: adMSCs) and a section of the corresponding fluorescence microscope images in higher magnification (b: HUVECs, d: adMSCs). Both detection methods are applicable. 2000 cells were printed each time and cultivated for 2 days, before staining was performed. a: PMT gain = 550; c: PMT gain = 450. The scale is in b and d 200 μm

3.5 Analysis of the Impact of Printing to MSCs by Examining Their Differentiation Potential

  1. 1.

    Analysis of differentiation potential is one procedure to demonstrate the impact of new methods or pharmacological reagents to primary cells. For differentiation experiments, transfer the desired volume of cell suspension to the prepared microarrays coated with collagen (see Note 10 ). Here, the same volume of suspended MSCs is once printed and once transferred via standard pipette to microarrays. Cultivate MSCs for 5 days until confluence was reached in each well, before culture medium is changed to the osteogenic, chondrogenic, or adipogenic differentiation medium. As a negative control, use undifferentiated MSCs cultivated in culture medium (Fig. 4).

     
  2. 2.

    Perform medium change every third to fourth day.

     
  3. 3.

    At the end of differentiation (after at least 21 days) wash cells once in 100 μl PBS and fix them in 100 μl paraformaldehyde solution (4.0% in PBS) for 30 min at 4 °C. Fixation procedure is completed with two further washing steps in 100 μl PBS. Thereafter, it is not necessary to perform all further steps under sterile conditions. Microarrays with fixed cells can be stored for month, if the surface is moist all the time.

     
  4. 4.

    To determine osteogenic differentiation, perform an Alizarin Red staining. Treat fixed cells with 70 μl Alizarin Red solution per well for 30 min at room temperature. Remove excess dye in extensive washing steps with 100 μl PBS each. Staining is followed by covering of cells with PBS and examination under microscope in brightfield mode.

     
  5. 5.

    Detect the differentiation toward chondrocytes by visualization of proteoglycans using Alcian Blue staining. Incubate fixed cells for 3 min in 70 μl acetic acid (3.0%) per well at room temperature, followed by a staining step with Alcian Blue 8GX solution for 30 min at room temperature. Finally, wash the cells three times in 100 μl acetic acid (3.0%) per well, cover them with PBS and detect bounded dye under microscope in phase contrast.

     
  6. 6.

    Visualize intracellular lipid droplets specifically using BODIPY 493/503 (4,4-difluoro-1,3,5,7, 8-pentamethyl-4-bora-3a,4a-diaza-s-indacene) fluorescence dye. Incubate fixed cells in 70 μl BODIPY working solution (5.0 μM in PBS) per well for 5 min at room temperature in the dark. Afterwards, wash the cells twice in 100 μl PBS per well and detect fluorescence of BODIPY under microscope using the fluorescence source and filter set for FITC.

     
  7. 7.

    Perform for samples visualized by fluorescence (at least for negative control and printed MSCs it is mandatory) an additional DAPI counterstaining afterward. Therefore, remove storage solution on the stained microarrays and stain cell nuclei by DAPI staining solution for 15 min at 37 °C in the dark. Wash stained cells twice in 100 μl PBS per well and image them using microscope equipped with fluorescence source and filter set for DAPI, e.g., BP330-385 (exciter filter), BA420 (barrier filter).

     
Fig. 4

After cultivation, ucMSCs underwent osteogenic (a), chondrogenic (b), and adipogenic differentiation (c). Positive controls (cells transferred to wells via pipetting and differentiated; first row) were stained red (extracellular calcium accumulation in a), cyan (extracellular proteoglycans in b) or green (intracellular lipid droplets in c). Negative controls (printed cells cultivated in culture medium; second row) did not show any staining (a) or only a weak staining (b and c); blue signals are DAPI counterstained cell nuclei. Printed cells (last row, marked by blue frame) are stained less strongly than positive controls but more than negative controls. The scale is in a, b 100 μm and in c 50 μm

4 Notes

  1. 1.

    Having 500–800 ml water in the flask helps to dissolve the powder relatively easily by swaying the flask. If 2 l medium or more are prepared, it is advisable to cover the flask with a piece of sterilized aluminum foil to prevent any kind of contamination during the long process of pumping through the pressure filter unit. After this filtration step, every further step dealing with cell culture has to be performed under sterile conditions in a laminar flow cabinet. To ensure sterility of the prepared basal medium, transfer a sample of up to 5 ml into a T25 cell culture flask and incubate it for at least 3 days at 37 °C in a humidified environment with 5% CO2.

     
  2. 2.

    According to manufacturer’s protocol Calcein-AM diluted 1:30 in PBS shall be added directly into the culture medium on top of the cells while a 1:10 dilution takes place. Following this protocol a high green-fluorescent background is resulting, due to the presence of serum in the culture medium. Therefore, we adapted the protocol to capture images. According to manufacturer’s protocol of another comparable viability staining assay the culture medium is removed.

     
  3. 3.

    Camera: Since the applications for microscopy in cell culture are varying, a camera preparing color images is used for documentation of bright field, phase contrast, as well as fluorescence samples.

     
  4. 4.

    Fluorescence microarray scanner: A variable pixel size is mandatory. A pixel resolution of 5 μm shows the best results, whereas a pixel size of 100 μm is inapplicable.

     
  5. 5.

    Since gentamycin is not stable, when it is frozen, it has to be added each time an aliquot of differentiation medium is thawed.

     
  6. 6.

    Instead of staining with Alizarin Red solution, a more specific and sensitive staining for osteogenic differentiation can be performed using Calcein, which shows a high affinity to accumulated calcium ions and is detected by fluorescence. Wash the fixed cell layer with 100 μl PBS and incubate it with 70 μl per well Calcein solution (5.0 μg/ml Calcein in H2O) at 4 °C overnight. Wash it twice with PBS and detect the fluorescence of bound Calcein under microscope using filter set for FITC.

     
  7. 7.

    Primary cells, like MSCs, undergo only a limited number of cell divisions. The cells go into senescence after several passages resulting in a growth arrest. They are metabolically active, but show functional alterations, for example in cell morphology, protein secretion, and differentiation [20].

     
  8. 8.

    Proliferation of HUVECs is limited much earlier. It is known that they change their physiology, beyond reaching a passage number of 5–7 [21, 22]. Furthermore, their cell growth is getting very slow. Therefore, all experiments with these endothelial cells are limited to passage 1–5.

     
  9. 9.

    Nanoprinter manufacturer analyzed the volume of 1000 water droplets for each pipetting tip individually using a pulse width of 50 μs and different frequencies and voltages. Here, the pipetting tip characteristics are: 1d = 0.0002 μl. This means that for pipetting a volume of 0.6 μl 3000 droplets are necessary. When 3000 cells shall be printed using a suspension volume of 0.6 μl, a cell suspension of 250,000 cells in 50 μl is used.

     
  10. 10.

    HUVEC show an enhanced growth on fibronectin, which can be used for coating the microarrays instead of collagen. For this purpose, sterile human fibronectin solution is diluted to prepare a 10 μg/ml working solution that is used for covering the microarray surfaces. After incubation at room temperature for 60 min, the solution is aspirated and the microarray can be used for printing. Fibronectin coating is also helpful, to differentiate ucMSCs. Differentiation of these MSCs is sometimes challenging, but using fibronectin coating leads to an enhanced differentiation potential.

     
  11. 11.

    If the coated microarray surface is moisturized, the monitoring of printing is not possible. Due to this, we developed a protocol for printing cells onto dry well surfaces, followed by the addition of culture medium 1.5 min subsequently [15], which also leads to highly defined circular spots.

     
  12. 12.

    In general, cell suspension can be printed to a variety of targets with different dimensions, e.g., cell cultivation well plates or chamber slides. Dimensions of each target have to be transferred to the working plate in the nanoprinter software, before the printing. Since this transfer takes some time, this step should be finished before the cells are prepared for printing. Furthermore, the spot layout of the target group has to be determined for each printing step, including their margins and the arrangement of blocks within the target group, as well as the number of droplets.

     
  13. 13.

    Before the printing can be started a z-measurement has to be performed to prevent a damage of the glass pipetting tip caused by differences in altitude of the microarray surface. Using the incubation chamber this is not possible, as the z-measurement is performed with a flexible needle that is applied next to the pipetting tip. Therefore, this measurement has to be performed with a dummy that shows the same high as the microarray inside of the incubation chamber exhibit. It is advised that this process is finished before the cells are prepared for printing.

     
  14. 14.

    Printing one spot per well, a position of the pipetting tip in the middle of the printing area is preferred. For example, the pipetting tip transfers a defined amount of the sample placed in well A1 of the donor template to the centre of each well of the microarray incubation chamber.

     
  15. 15.

    The nanoprinter itself can be challenging, but following some rules the printing process is easy and reliable. If there are bubbles in the tubes, empty and fill the tubes again. It is highly recommended that inner parts of the pipetting tip are always wetted, so use the manufacturer’s cap. If the nanoprinter is not used for a longer time period, wash the tip once a week. An additional washing step before printing of cells with 2-propanol is preferable but not mandatory, since 2-propanol can be challenging for the wetting of the pipetting tip walls.

     
  16. 16.

    To prevent settling and aggregation of cells, cell suspension is transferred to donor template directly before the printing. Due to this, the sample is additionally mixed between the printing steps, and in each printing step only a small number of spots is produced (2–4). HUVECs tend to settle and aggregate very fast, making this procedure necessary for this cell type. If more than four spots are produced with the same pipetting tip filling, the dosage of cells is not reproducible. Increasing the viscosity of the cell suspension can be used to prevent this [9, 23].

     
  17. 17.

    The z-position (high) of the pipetting tip—target distance during the spotting and the move—is relative to the z-position of the target microarray identified during z-measurement, making the z-measurement with a dummy very important. The printed spot is exactly positioned at the defined printing area, when the target distance is very low. An increase of the distance results in a shift of the printed spot, especially in chambers made of plastic. Therefore, the pipetting tip always extends into the well. Caution: Damages of the pipetting tip are possible, when the microarray is positioned wrong on the target side, or other chamber systems are used without changing working plate settings!

     
  18. 18.

    It is of note that within the first hours of monitoring cell growth, the light may change a bit in brightness and the focus position may shift slightly. It is highly recommended to correct both parameters in the first hours to ensure a high quality time-lapse video.

     
  19. 19.

    Online monitoring with incubator microscope can be challenging because of light scattering effects. The smaller the diameter of the well, the higher the curvature of liquid meniscus and the higher the light scattering. Therefore, online monitoring of cells is preferred in slides with larger well dimensions (e.g., chamber slides from Nunc or ibidi). Besides this, a fogging of the microarray backside and the inner surface of the lid are challenging. Let the liquid inside the wells equilibrate to the incubator’s temperature. It can be helpful to wipe the microarray and lid clean with a tissue moistened with 2-propanol.

     

Notes

Acknowledgment

This work was supported by the BIOFABRICATION FOR NIFE Initiative, which is financially supported by the Lower Saxony ministry of Science and Culture and the VolkswagenStiftung. NIFE is the Lower Saxony Center for Biomedical Engineering, Implant Research and Development in Hannover, a joint translational research centre of the Hannover Medical School, the Leibniz Universität Hannover, the University of Veterinary Medicine Hannover, and the Laser Center Hannover.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Technical ChemistryGottfried Wilhelm Leibniz Universität HannoverHannoverGermany

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