Next, the tubular scaffold was connected to a syringe pump system using a polydimethylsiloxane (PDMS) microconnector for long-term cell culture. Immunofluorescence analysis of newly synthesized type I collagen (magenta) in cell scaffolds with an anti-mouse type I collagen antibody (converted from Alexa 594 red to magenta). In this field, the development of engineered blood vessels is one of the key challenges for the successful clinical use of tissue engineering constructs [1].
Only the natural diffusion of the immersion method could cause uneven cell differentiation and proliferation beyond 150 µm wall thickness. The negative pressure of the microconnector of the syringe pump connected to the inlet breathed the boom into the outlet. The elasticity of the calcium alginate scaffold and tigon tubing connected to the syringe pump allowed secure connection to the PDMS microconnector for eleven to fourteen days.
The syringe pump supplied cell medium into a central channel of the tubular scaffold through the PDMS microcoupler. In order to minimize the effect of medium diffusion from outside the tubular scaffolds, two-layer calcium alginate tubular scaffolds were rapidly generated by a bi-coaxial laminar flow generator (Fig. 1). This hydrogel outer-only layer acts as a barrier to prevent cell medium outside the outer layer from diffusing into the cells in the inner layer.
Cell medium was pumped through the middle channel of the scaffolds with three conditions: cardiac mimetic pumping, continuous pumping, and non-pumping.
Fibroblast cell preparation
In this study, we pumped cell medium into cells in a tubular scaffold using a polydimethylsiloxane (PDMS) microconnector for approximately twelve days. The PDMS microconnector consists of one outlet for the scaffold and one inlet for the syringe pump. After twenty days of cultivation, cultured scaffolds of these three conditions were analyzed and compared in terms of cell viability, cell proliferation, intercellular junctions, and secretion of ECM material.
Subculture and harvest
Sodium alginate and CaCl 2 preparation
PDMS double-coaxial laminar flow generator
The three inlets were heat-drawn with a PC-10 puller (NARISHIGE Group, USA) to be automatically pulled to achieve an inner diameter of about 200 µm with a tapered shape prior to mounting on the PDMS block (Fig. 1C). The two PDMS blocks were fabricated by coagulating the silicone elastomer blend (Dow Corning Corporation, Midland, USA) on a partially drawn glass capillary. After the heat gelation process, the partially drawn glass capillaries were replaced with the inlet glass capillaries, the intermediate glass capillary and the outlet capillary for a secure connection.
The inlets, intermediate glass capillary, and outlet are assembled using PDMS block 2 and attached to the microscope glass. -d) Microscopic observation of the intersections between the inlet openings and the intermediate glass capillary and between the intermediate glass capillary and the outlet opening. e) The concept of stage design. A bilayer NIH/3T3 calcium alginate hydrogel scaffold was continuously generated in a PDMS dual coaxial laminar flow generator before external gelation in CaCl2 was completed. f) The scaffold structure consisted of one outer layer of calcium alginate and one inner layer of NIH/3T3 calcium alginate.
The cavity was formed by crosslinking activity at the intersection between inlet A and inlet B in the generator.
Two-layered tubular scaffold formation
PDMS connector
At the inlet of the PDMS connector, a plastic tube with an inner diameter of 640 μm (Tygon E-Lab extension tube, Harvard Apparatus, USA) was inserted, which was connected to a syringe pump. Due to the negative pressure from the attached syringe, the PDMS connector absorbed a seven-centimeter-long scaffold into the socket (Fig. 3. a). We usually suction the tubular scaffold to a position within about 3 cm of the exit point.
Medium pumping configuration
Live/Dead staining
Immunocytochemistry for cadherin
Immunocytochemistry for newly-produced type I collagen
Microscopic imaging and analysis
Statistical analysis
Two-layered cell-laden tubular scaffold
Disconnection test
Twenty microliters per minute, the volumetric flow rate in human retinal blood vessels, delivered up to days in a continuous pumping condition and days in a heart-mimicking pumping condition. There were significant differences between continuous pumping and cardiac-mimicking pumping conditions (p < 0.05) when tested at a flow rate of 20 µL/min and 50 µL/min (p < 0.01) at a flow rate of 30 µL/min.
Proliferation in the three pumping conditions
Do not leave up to 15 days. in contrast, the other two pump conditions showed smooth distribution until day 11. Number and area of the green gradually increased. No major difference between the continuous pumping condition figures and the cardiac mimicking pumping condition figures was recognized. Since the disconnection, the disconnected scaffolds were immersed in medium only until day 20 without any medium pump.
Compared with day 13 and day 20, the outer living cells of the two pumping conditions decreased and the dead cells suddenly increased. Live cells were stained green by calcein-A. 7 a - c shows a fluorescence-stained image of a cell cluster in the pumped heart-mimicking scaffold at day 7. To quantitatively analyze live stained cells, their volumetric distribution was investigated using a three-dimensional analysis function of ImageJ. Based on this assumption, histograms of cell groups were plotted, as shown in Fig.
Up to day 5, all three pump conditions showed a similar number of the smallest cell group with a population of 1 ~ 3 cells. The population of the largest cell group of the non-pumping condition was also much smaller since day 7 than that of the other two pumping conditions. By day 7, the most volumetric cell group of the two pumping conditions with a population of 37 ~ 39 cells had appeared and survived to day 20, the last day of observation.
In the case of the non-pumping group at day 11, the most volumetric cell group with a population of 19 ~ 21 cells had emerged and survived up to day 20. Averaged over all three pumping conditions and the entire culture time, a cell group had 7, 80 cells and its standard deviation is 15.47. Since day 7, the total volume of the non-pumping condition has decreased, but that of the other two pumping conditions has increased up to day 13.
As of day 7, the volume contribution of the large group was over 50% and increased after disconnection. On day 7, the most volumetric cell group in the two pumping conditions was almost twice as large compared to the no-pumping group, as shown in the figure. After that, the volume of the most volumetric cell group in the two pumping conditions gradually increased around disconnection and then slightly decreased on day 20.
Cadherins in the two-layered cell-laden scaffolds
11 d–f, NIH/3T3 seeded in a conventional two-dimensional culture dish, cadherins in cell-laden scaffolds are more widely occupied. NP HBMP CP NP HBMP CP NP HBMP CP NP HBMP CP NP HBMP CP NP HBMP CP NP HBMP CP.
Type I collagen in the two-layered cell-laden scaffolds
The outer cellless calcium-alginate layer made it possible to observe only effects of the pumped medium. The generated scaffolds consisted of the core channel, the cell-laden layer and the calcium-alginate hydrogel sole layer from inside to outside (Fig. 4). Considering distribution of the proliferation and newly secreted ECM (Fig. 6 and 12), the calcium-alginate hydrogel sole layer caused the outside medium to diffuse into the cell-laden layer.
The food dye injected from the syringe pump flew to the end portion of the two-layer tubular scaffolds through our microcoupler without any leakage (Fig. 3). In the culture medium pumping experiment, the heart-mimicking pumping condition was sustained for 14 days and the continuous pumping condition for 12 days. These long safe connection times can support favorable conditions for proliferation, elongation and differentiation of the seeded cells such as human umbilical vein endothelial cells (HUVECs).
In the present connection, the tubular scaffolds are absorbed up to 3 cm from the outermost location of the connector exit. We pumped cell medium under three conditions: heart-mimicking pumping condition (20 µL/min in flow rate, 1 second in period and 50% in pulse width), continuous pumping condition (20 µL/min in flow rate of flow) , and non-pumping condition. No significant difference was perceived between the heart-simulating pumping condition and the continuous pumping condition.
From day 0 to day 5, the total volume of the three scaffolds was not significantly different, but the distribution of the small group and the medium group is so different (Fig. 9). On day 7, the total volume of the unpumped scaffold began to decrease, but that of the other two pumped scaffolds began to increase (Fig. 9). Confocal microscopy images ( Fig. 6 ) and volumetric analysis of the cell cluster ( Figs. 8 – 10 ) showed that the large cluster appeared in the two pumped scaffolds at day 7.
The signal of the type I collagen became stronger compared to the previous day (Fig. 12). Up to day 13, the live cell images of the two pumped scaffolds showed gradually increasing proliferation (Fig. 6). After the disconnection, the most volumetric cell groups of the two pumped scaffolds slightly decreased in volume (Fig. 8), even though their total volume decreased suddenly (Fig. 9).
