Indirect Measurement of Cell Loading in Hydrogel Bioinks During Handling and Preparation of 3D Models

Introduction: : We present an inverse engineering method of estimating physical forces within cell suspensions in 3D scaffolds during fabrication using
fluorescent, lipid-coated microbubbles (MBs) filled with an inert fluorocarbon gas. MBs have the property of deforming and rupturing under significant mechanical pressure. Here, a two-step protocol is described for estimating processing forces in cell-laden hydrogels when cells possess a spherical form. We used fluorescent lipid shell MBs of a C4F10 gas core. The leaked fluorescence from the MB shell was then detected and quantified. Additionally, we investigated the relationship between the mechanical properties of the MB shells and hydrostatic pressure using ultrasound imaging. We examined hydrostatic pressure-dependent changes in MB stability and fluorescence leak after rupture when MBs were in I) PBS or II) encapsulated within a GelMA scaffold. We quantified and compared the fluorescence intensity of the ruptured MBs for varying pressures to form calibration curves. The curves were then used to quantify the physical forces around biological cells in similarly made hydrogels.

In this short communication, the compressibility characteristics of the MBs were applied to estimate the forces acting in cell-laden models during the fabrication and handling processes. MBs encapsulated in hydrogels were subjected to hydrostatic pressure for a specific period, and the calibration curves generated were able to estimate the pressure around each MB. We further studied the MB response via conventional ultrasound imaging and theoretical modeling. This work offers a simple method of estimating physical loads onto biological cells during extrusion, injection, and other forms of fabrication.

Materials and

Methods: : MBs were freely suspended in PBS, loaded into 20 ml syringes, and connected to a universal testing machine (Instron, MA) until specific pressure values were reached (0-500 kPa) with fixed time points (1min, 5min, 25min). The MB-PBS solution (50 and 100 k/mL) from the different experimental groups was filtered (0.22 μm) to separate the still intact MBs from the ruptured fluorescent micelles (size < 200 nm). 100 μl of the solution was then added to 96-well plates, and the FITC intensity was quantified using a plate reader. The supernatant fluorescence was related to pressure, and calibration curves were plotted. For the second experimental setup, MBs were encapsulated in GelMA hydrogels (5%, 7%, and 10% w/v), which were cylindrically shaped (5mm radius, 5 mm height), and the measurement of their responses to hydrostatic pressure was carried out. The extent of rupturing was quantified by measuring the supernatant fluorescence using a plate reader for the solutions (PBS) in which the MBs and hydrogels were placed. The syringe was in direct contact with the load cell, the fluid was in free suspension within the syringe, and a solid needle plug was connected to the end of the tubing to make a closed loop. Pressure build-up inside the syringe led to an eventual rupture of the MBs, releasing fluorescently tagged lipid micelles into the free solution. The procedure was repeated for two MB densities (50 and 100 k/mL), which were selected based on the practical limitations. Statistical analysis was performed using two-way ANOVA.

Results, Conclusions, and Discussions:: We quantified the fluorescent signals of the MBs under hydrostatic pressure when it was applied in our custom-made pressure setup.
Hydrostatic pressure on the gas-filled MBs suspended in PBS for two MB densities (i.e., 50 and 100 k/mL) was used for forming the calibration curves. As the pressure increased from 0 kPa to 500 kPa over 25 min, a non-uniformity of the size distribution was observed. The fluorescent images of the MBs in a 100 μl solution showed that the MBs significantly deformed under hydrostatic pressure until a threshold pressure was reached (~ 250kPa), after which the MB count indicated rupture. There was an almost linear decrease in fluorescence intensity for the intact MBs. The MBs at 40 kPa, 1 min condition showed the highest fluorescence compared to MBs experiencing a 500 kPa pressure for over 25 mins. The fluorescence of the ruptured membrane/vesicles in the supernatant solution was then quantified and normalized to the case of zero-pressure conditions. The recorded intensity of the supernatant solutions showed a relatively linear increase up to 200 kPa over 25 min, after which there was an exponential increase in the readout versus the pressure level and time. The ruptured MBs or supernatant solution fluorescence data provide a quantifiable relationship to the pressure level applied to the group of MBs in the solution. This correlation was further studied in our theoretical simulations.

Pressure changes can affect the size of a contrast MB by mechanical compression and gas efflux. The latter process depends on the permeability of the encapsulating layer. Therefore, gas exchange plays a vital role in determining the size of the MBs and their mechanical properties. This information can be correlated to pressure effect on cells under similar conditions. The size and stability of the gas change as a function of the local hydrostatic pressure. Thus, compared to invasive techniques such as atomic force spectroscopy, on-demand MB dissolution can be used as a noninvasive real-time method to estimate forces within 3D environments, which can then be correlated with pressure effects on cellular markers.

Acknowledgements and/or References (Optional):: Dr. Amir K Miri acknowledges NIH-R01-DC018577 and NSF-EBMS-2243506 grants for this research project. The authors acknowledge the contributions of Dr. Elvan Dogan, Mrs. Ayda Pormoustafa, Ms. Leila Donyaparastlivari, Ms. Vidhi Patel, Ms. Diana Saad, Dr. Alexander Buffone Jr., Dr. Rajarshi Chattaraj, and Dr. Amir K Miri.

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