bioprinting of 3d convoluted renal proximal tubules on perfusable chips
Drug screening, disease modeling, and final kidney organ engineering need to reproduce the dimensional model of the kidney tissue of the human response.
Here, we report a biological printing method used to create a 3D human kidney proximal tube in vitro that is completely embedded in the extracellular matrix and stored in a perfusion Tissue Chip, they were allowed to last for more than two months.
Their complex tubular structures are limited by near-curved epithelial cells and actively perfusion through an open cavity.
The 3D nearly curved tubes designed on these chips have significantly enhanced epithelial morphology and functional properties relative to the same cells grown on 2D controls, whether or not perfusion.
After the introduction of renal toxin csa A, the epithelial barrier was destroyed by dose
Our biometric printing method provides a new way to programmable on-demand manufacturing of advanced human kidney tissue models.
The extracellular matrix is composed of gelatin and fib.
To prepare ECM components, 15 wt/v % gelatin solution (
Type A, 300 bloom of pig skin, Sigma)
First produced by adding gelatin powder to the warm solution (70u2009°C)of DPBS (
1X Dulbelco phosphate buffered saline without calcium and magnesium).
By stirring for 12 µh at 70 °c, gelatin is allowed to dissolve completely and then the pH is adjusted to 7.
5 Use 1 sodium hydroxide m.
The solution is sterile filtered and stored in an equal sample at 4 °c for later use in casting ( 1 h in the oven and stored at room temperature before use.
3D PTs mapping within the perfusion chip requires a combination of casting ECM and printing unorganized ink.
First, an ECM solution was created by binding 10 mg/ml of FIB, 7.
5 wt % gelatin, 2. 5 mM CaCl and 0. 2 wt% TG.
The solution is then balanced for 15-20 minutes at 37 °c and then used to improve the optical clarity of the ECM.
Next, the solution is quickly mixed with the coagulation in a ratio of 500: 1, resulting in a final concentration of 1 u/mL.
At 37 °c, the cellulose was originally aggregated into the cellulose gel within 2 min.
Therefore, after mixing with the thrombosis, the ECM solution must be immediately cast at the bottom of the perfusion chip.
It is then allowed to dry slightly under nitrogen to form a flat surface.
The fugitive pludonic F127 ink (
Heparin with 100 u2009 U/mL)
Printed on the underlying ECM layer in the form of intricate films (tubule)
Use the cone 200 u2009 μm nozzle.
Custom Python script (MeCode)
Used to specify tool paths in G-code.
After the unorganized ink is printed, the metal hollow filling inserted through the silicone gasket is written off to contact the printing ink directly.
Then, by casting the ECM solution onto the printing tube, as previously described, a top-layer ECM is formed within 1-2mm of the height of the gasket wall.
If cells, such as HNDFs, are integrated into the ECM ()
, They are mixed directly after the balance period, before the coagulation mixing and subsequent casting.
After the ECM layer at the top is cast, the structure is covered by a slide to prevent evaporation or contamination and is maintained for 1 hour at 37 °c to allow the termination of the cellulose polymerization, TG cross-connects the network.
The structure is then cooled to 4 °c 15-20 min to liquidate the printed unorganized ink, which flows out of the device using cold battery media, leaving open pipes, these pipes are embedded in the outer cell space of the cell as an ideal tubular network.
Using this method, we also generate a 3D architecture in one layer. by-
Layer build sequence.
For example, three separate layers
The layer structure shown in is built using the modified print protocol, which contains the materials and methods discussed earlier.
After printing the first tube with unorganized ink, cast a layer of ECM on the print and allow the gel at 37 °c for 20 minutes, then print the next near-curved tube layer at the top of the nearest gel layer with unorganized ink.
This continuous construction introduces 3D geometry and allows for independent evacuation of all passages after construction. Aqueous-
The risk-based reactor dyes are infused through the Channel and are stimulated by ultraviolet rays for visualization.
To complete the 3D tissue chip assembly process, place each PT structure on the machined stainless steel base and place a thick acrylic cover on the top.
The cover and base are clamped together by four screws to form a seal around the printed silicone gasket.
Next, sterile 2
Stop hose (PharMed BPT, 0.
25mm inner diameter)
Fill the medium and connect to the outlet of the sterile filter attached to the 10 ml syringe barrel (EFD Nordson)
As a media reservoir. PTEC media (
ATCC formula plus 1% FBS, 1% aprotinin and 1%anti)
This has been balanced in the incubator at 37 °c> 3 u2009 h, 5% CO is added to the medium container, the pipe from the medium container is connected to the outlet of the chip (
Metal hollow irrigation needle).
The syringe is then used to apply a slight pressure on the medium in the barrel, forcing it to enter and fully fill the connected pipe.
Before connecting the pipe to the circuit, filling the pipe with media can prevent bubbles from entering the system.
In order to complete the perfusion circuit, the silicone pipe from the reservoir is connected to the inlet metal irrigation on the chip to cancel. Hose pinch-
An off clamp has been added at the inlet and outlet of the perfusion chip to prevent uncontrolled flow when disconnected from the peristaltic pump, which may damage epithelial cells or allow bubbles to enter the system.
Media containers are always balanced with atmospheric conditions in incubators through Sterile filters at the top of media containers.
Human eternal life PTECs (
RPTEC/TERT1, atcc crl-4031)
Culture is carried out according to the instructions of ATCC and used until all PT model studies through 20.
For gene expression analysis, human primary RPTEC (Cell Science)
Eternal PTECs (RPTEC-TERT1, Evercyte)and A498 (ATCC HTB-44)
Use and culture renal tumor cells according to supplier instructions.
Human neonatal dermal fiber cells (HNDF)
Green fluorescent protein expressed (Angio-Proteomie)
Train according to supplier\'s instructions and use to pass 15.
Human primary RPTEC (Cell Science)
Eternal RPTEC-TERT1 (Evercyte)and A498 (ATCC HTB-44)
Kidney cancer cells grow in 96-
According to the supplier\'s instructions, collect the orifice plate after the third day
Fusion of the medium was replaced by 100 µμ l/well of 1x RNA lysate mixture (
Quantitative Gene sample processing kit (QS0101).
Then mix 40 μ l lysate with mRNA
Capture magnetic beads Group (
Panomics QuantiGene Plex Set 12631, catalog number 312631)
, Incubation overnight, branch DNA amplification processing, and analysis according to the manufacturer\'s instructions (
Panomics Quantitative Gene complex analysis kit (QP1015).
PPIB probes are used for normalization as housekeeping genes.
Fluorescence intensity (FI)
The data are shown as mean and standard deviations for 3 biological repetitions.
Within 25 days of cell inoculation, culture medium perfusion fluid was collected from tubular cells and stored at-80 °c before analysis.
For the cell factor spectrum, unfreeze the culture medium on ice and dilute 2x in sample dilution buffer (
BioRad company directory M60-009RDPD)
And analyzed the Luminex technology.
Plex Pro™Man IL-6 (Set 171BK29MR2), IL-8 (Set 171-BK31MR2)and MCP-1 (Set 171-BK36MR2)and the Bio-
Plex 200 system (BioRad)
According to the manufacturer\'s instructions.
The data report is the mean cytokines concentration and standard deviation of the three reports of the technology.
Before cell loading/sowing, each 3D PT builder was infused with PTEC medium in the incubator for several hours. PTECs (
Digest and condense from their culture plate ~ In the medium of 2 × 10 cells/mL.
Then the cell suspended matter is loaded into the perfusion chip through the outlet ().
The loaded structure is placed horizontally in the incubator for several hours and flipped 180 ° over multiple and a half hours
Intervals are made once an hour to allow the tubular walls to be evenly sown and then incubated overnight in tubes that do not flow.
The next day
Under the action of gravity, the adherent cells are discharged from the tubular cells.
Then start pouring the fresh medium, the remaining cells start to gather, and then grow from these colonies ()
Until they reach fusion about 3 weeks after sowing ().
In the growth stage, PTEC medium prepared according to the ATCC guidelines plus 1% aprotinin (
Used to slow down the degradation of ECM)
1% fetal bovine serum (FBS)
1% of antibioticsantimycotic (Gibco).
After maturity, FBS are removed and PTECs are packaged into a tight epithelial single layer (). At Day 1 post-
At sowing time, PTECs were exposed to continuous one-way flow at 1 μ l/min, equivalent to the shear stress that changed between 0. 1 and 0.
5 dydynes/cm, depending on the cross section of the fallopian tube.
In the closed loop, the medium is delivered through the peristaltic pump and replaced every 2 days.
For the printed 3D PT model and 2D controls, the protein uptake was evaluated.
The first control consists of PTECs grown on tissue culture plastics, and the second control consists of PTECs grown on our ECM.
In each case, PTECs can mature in serum-free medium.
Human serum protein and collagen (HSA-
Of Abcam ab8030)
Suspended in a PTEC medium of 50 μg/mL.
All samples with human serum protein-
Perform 2 Thanh in their medium (
In the case of perfusion, perfusion is performed through an open cavity).
After exposure, wash all samples with 3 times volume and then digest with 10 times pancreatic enzymes to collect individual cells.
Fixed and re-stained cells with megalin\'s first and second resistance (
List specific antibodies used).
The cells from these samples and nude cells were analyzed by flow microscopy (Bd lsr fortssa)
Data collected from 50%, 70%, 90%, 2 × 10 min 100%).
Then put the sample into the propylene oxide (EMS)
1 u2009 h, incubated overnight in a mixture of 1: 1 of propylene oxide and TAAB Ethernet (
The next day, the sample was embedded in TAAB Epon and aggregated for 48 h at 60 °c.
Ultra-thin slices (about 60u2009nm)
Cut on the Reichert super cold machine
S slicer, placed on a copper mesh stained with lead citrate, examined in a JEOL spirex transmission electron microscope, images recorded with an AMT 2k CCD camera.
Perform Image analysis using Image j software.
For scanning electron microscopy (SEM)
, PTECs poured in 3D were fixed for 1 hour with 10% buffered formaldehyde.
Cut the sample into slices (~1u2009mm thick)
Expose cells that limit the open cavity.
Wash the fixer away using PBSx2 and then dehydrate in different grades of ethanol (20u2009min each;
30% 50% 70% 90% 100% × minutes).
Then put the sample into 50% ethanol and 50% methyl diazol (HMDS)
30 minutes, then 100% HMDS 3 min x 30 min.
All steps are performed in closed and sealed glass containers.
After final washing with HMDS, take out the sample and put it into the open container under N in the fume hood for drying.
The dry samples are mounted on an aluminum pin bracket using a conductive carbon tape, with a gold coated spatula, and are imaged with Tescan Vega SEM.
Immune staining and co-focusing microscopy are used to evaluate the localization of proteins in 2D and 3D PTEC models.
Before immune staining, each build was washed with PBS and then fixed for 20 minutes to 1 hour with 10% buffered formaldehyde.
Use several washes in PBS to remove the fixer for a few hours, and then use 1 wt % of bovine serum protein for the night (BSA)in PBS.
An antibody to a cell protein or an interested biomarker listed in 0 solution incubated with the build for 1 day. Poser and 0 for 5 wt %.
X-125 weight %100.
Use the washing steps for the solution of PBS or 0 to remove the uncombined one-antibody. Poser and 0 for 5 wt %.
X-125 weight %
100 place for 1 day in PBS.
The dilution listed in the 0 solution of the secondary antibody was incubated with the build for 1 day. Poser and 0 for 5 wt %.
X-125 weight %100 in PBS.
The sample is reversed.
DYE 2 Thanh with NucBlue or ActinGreen before imaging and then wash in PBS for 1 day.
Phase contract microscopy using the inverted Leica dm il range with a target range of 1. 25X to 40X.
The co-focal microscope was performed using an upright Zeiss m6 710, with a water immersion target ranging from 5X to 40X, using a spectral laser of 405, 488, 514, 561 and 633 nm wavelengths.
Image reconstruction of Z-
Stack is using z-
Projection function with maximum pixel intensity setting.
Any increase in brightness is in the entire z-Projected image.
3D image reconstruction and rotating film ()
Execute with Imaris software.
New cell Smart (Lonza)
In the incubator system for capturing time-lapse imaging ().
Use a custom MATLAB script to perform image analysis of diffusion permeability quantification using previously reported methods.
TEM image analysis using image j software to measure cell height (≥u200950)
Density of microfluff (≥u200925)
Length of microfluff (≥u2009150)
There are at least 3 independent samples per condition.
The data is expressed as mean ± standard deviation.
Statistical analysis is performed using MATLAB and statistical significance is determined with the value of p