# Biofab Box: oxygen-model verification protocol

Run `67f4272f0fb6bbf3`. Proposed engineering study. No fabrication or biological verification has occurred.

## Decision and hypothesis

Which printed gel thickness should we test first, and does measured oxygen agree with the transport prediction?

Compare three printed cylinder heights at the same material composition and initial cell density. The simulator predicts a spatial oxygen field and the oxygen near the center of the gel floor. Measure oxygen directly to test that prediction. Viability and secreted-function assays are separate secondary observations.

## Reference system and evidence

Reference cells: HepG2/C3A; this is an engineering model, not a primary-human-hepatocyte drug-safety assay. Bhise et al. (2016, DOI 10.1088/1758-5090/8/1/014101) modeled printed hepatic constructs using Michaelis-Menten oxygen uptake. Its numerical assumptions supply the high-uptake reference here. Our static well and uniformly cell-loaded cylinders differ from its perfused spheroid system.

The proposed material is GelMA. Select the actual product/lot and a lab-qualified preparation and curing procedure before biological work. The source paper used a particular GelMA/photoinitiator/light setup; its lamp power is not a transferable irradiance setting. Preserve material, initiator, wavelength, measured irradiance, exposure, temperature and elapsed loading time in the run record. The actual extrusion pressure/speed and cure exposure are outputs of qualification, not guessed values in this export.

## Fabrication targets generated from this simulation

| Condition | Diameter mm | Height mm | Gel µL | Cells at target density | Medium µL |
|---|---:|---:|---:|---:|---:|
| H0.2 | 1.60 | 0.20 | 0.402 | 4021 | 63.94 |
| H0.6 | 1.60 | 0.60 | 1.206 | 12064 | 63.13 |
| H1.0 | 1.60 | 1.00 | 2.011 | 20106 | 62.33 |

Modeled well: radius 3.2 mm, liquid surface 2 mm above its floor. Volumes account for gel displacement. These are design assumptions, not confirmed plate specifications. Measure the actual well, deposited shape and medium depth; rerun with those dimensions if they differ.

## Materials, equipment and responsibilities

Recovered September 1 founder photo inspected September 25: label says Creality CR-10, rated build size 300 x 300 x 400 mm. This identifies the donor printer; current motion, controller and conversion status require physical qualification.

Required: the converted printer and local controller; qualified sterile fluid path, syringe and nozzle; registered plate carrier; microscope or profilometer with calibrated scale; balance suited to the dispensed quantity; the selected gel and cure apparatus; authenticated cells with a current contamination check; qualified culture lab and its cell-lot SOP; incubator; calibrated oxygen microsensor or compatible spatial oxygen-imaging method; temperature measurement; cell-count, viability and chosen secretion-assay equipment. A trained lab operator owns aseptic handling and culture, while Biofab owns the calibrated fabrication steps and logs.

## Phase A: qualify the printer with cell-free material

1. Document the real printer model, axis travel, controller, syringe geometry, nozzle dimensions and plate SKU. Register the carrier, establish a dry datum, and verify nozzle clearance across all used wells. Run the planned motion without dispensing, with the nozzle safely clear. Log offsets and travel limits.
2. Verify balance/volume measurement resolution for the smallest target. If individual deposits are below measurement resolution, weigh repeated deposits together to estimate mean delivery, and measure individual shapes optically. Measure the material density at the working temperature to convert mass into volume.
3. Calibrate command-to-volume using the actual material, nozzle and temperature across the required dose range. Account for startup lag, backlash, dripping and reservoir settling. Record fitted slope/intercept, residuals and the command range. Choose extrusion rate and motion settings from this calibration; no universal settings are presumed.
4. Produce at least ten cell-free deposits per geometry on each of three independent setups. Measure footprint, height and delivered quantity after curing and equilibration. A proposed release target is mean volume/height error within 10% and within-setup CV at most 10%; these are engineering acceptance choices, not measured capabilities. Re-run the model at observed dimensional extremes before deciding whether 10% is adequate.
5. Qualify the selected gel/cure combination using cell-free specimens first, then the lab's cell-compatibility procedure. Confirm shape retention over the intended observation window and record swelling. Cure and shear damage are not included in the oxygen solver.

## Phase B: characterize inputs before testing predictions

6. Calibrate the oxygen instrument at the experiment temperature and medium using its manufacturer's procedure. Record calibration points, uncertainty, response time and spatial footprint. Measure surface oxygen in matched cell-free wells. If the instrument cannot resolve the planned point or relevant gradient, change the sampling geometry and model before collecting comparison data.
7. Determine the cell lot's oxygen-consumption range independently of the comparison cylinders, and determine or justify the material's oxygen diffusivity. Record these as calibration data. The current broad literature envelope is for planning; it is not evidence that the prediction has passed. Freeze revised inputs, source hashes and predictions before opening the verification measurements.

## Phase C: fabrication and lab verification

8. Use three independently prepared batches on separate days. Each batch has three technical printed wells per height and endpoint, one cell-free gel per height, three unprinted handling controls and three medium blanks. The exported plate map allocates separate 24-hour and day-7 endpoints so destructive assays do not return to culture. Each batch also has a companion QC plate with three live-reference, three dead-reference and three matrix-blank wells per endpoint. There are 108 experimental/control wells plus 54 assay-control wells across three batches. Batch, rather than well, is the independent unit; this is an exploratory engineering design, not a powered assay-validation study.
Handling controls use the middle geometry (0.6 mm), 1.206 µL gel and nominally 12064 cells per well. Use the same batch, cell density, material composition, curing and total handling time as the printed condition; form the cylinder in a qualified mold without passage through the printing nozzle. Measure its actual geometry. This tests the contribution of dispensing shear/handling and is not a universal healthy-cell reference. Cell-free controls use each matching gel geometry and cure; medium blanks contain no gel or cells.
Prepare companion live/dead assay references using the selected assay's validated reference procedure in the matched material/assay matrix and working volume; verify their reference state independently. Do not assume that an unprinted construct is fully viable. Matrix blanks contain no cells. Record the exact reference preparation and lot in the assay SOP before beginning. Any optional secretion assay requires its own kit standards, QC and matrix blanks on a separately documented assay plate.
9. Prepare cells using the selected cell supplier and lab SOP. Record cell identity, passage, viable count, medium composition and time outside controlled culture. Prepare one uniform cell/material mixture per batch at the exported target density. Verify density independently. Use the calibrated dispense-to-volume mapping, approved material temperature, nozzle, cure and operator sequence. Record deviations instead of hiding failed prints.
10. Measure actual gel geometry, add the calculated medium volume, and verify liquid height. Transfer to the qualified culture environment. Use the lab's established temperature, gas and media-maintenance SOP for the actual cell lot, and record it. Those environmental conditions must reproduce the measured boundary concentration used by the model.
11. Acquire oxygen near the modeled center/floor sampling location and at the liquid surface, with measured spatial coordinates and acquisition uncertainty. The output location is the center of the first finite-volume cell, not an exact surface measurement. Require a stable reading before comparing to steady state: a proposed rule is less than 1% change over ten minutes. Record time-to-stability rather than treating the solver as a time-course prediction. If cells aggregate, proliferate, detach or substantially change geometry, the original uniform-density prediction no longer applies.
12. Collect viability and morphology at the separate endpoints using the lab's validated assay and blank/positive controls. If liver-function observations are included, choose an assay and record sampling interval, media exchanges, dilution and cell normalization. Albumin/urea/CYP measurements are not predictions from this model. Drug challenges require a later, separately qualified study and are not part of this oxygen verification.

## Comparison and decision, declared before results

13. Compare measured oxygen with the prediction at the same spatial location, geometry and surface boundary. The broad corner envelope is an uncertainty display. For the calibrated pilot, proposed numerical acceptance is mean absolute batch error no greater than 20 µM and abs(mean batch error) no greater than 10 µM, with measurement uncertainty no greater than 5 µM. These tolerances are proposed engineering criteria and must be frozen before running the lab study. For each geometry and endpoint separately, calculate each technical well's error as measured minus predicted oxygen, average those errors within each batch, then calculate MAE and absolute signed mean across the three independent batch errors. Report missing/excluded wells and their predeclared reasons; do not pool geometries or endpoints. Report every batch and the thickness ordering. Do not count technical wells as independent batches or tune parameters on the verification wells and call the same wells a validation.
14. A disagreement triggers review of measured geometry, cell density/uptake, medium depth, boundary oxygen, sensor location, gel diffusivity and the uniform-cell assumption. Preserve the failed prediction and label the next run as a revised model. Agreement verifies oxygen transport only within this tested system; it does not establish living-tissue performance, liver function or drug-safety accuracy.

## Execution status

This export is a complete prospective study plan plus fabrication targets. It is not calibrated G-code. Machine dispatch remains disabled until the following facts are supplied and verified:

- CR-10 actual motion envelope, controller and conversion qualification
- syringe/nozzle and extrusion calibration
- plate SKU and measured well geometry
- cell lot and lab-approved culture SOP
- gel lot/formulation and calibrated photocuring
- oxygen measurement method and calibration
- qualified lab access

## Sources

- A liver-on-a-chip platform with bioprinted hepatic spheroids: https://escholarship.org/content/qt9f546248/qt9f546248.pdf
- Analytic Models of Oxygen and Nutrient Diffusion, Metabolism Dynamics, and Architecture Optimization in Three-Dimensional Tissue Constructs: https://pubmed.ncbi.nlm.nih.gov/26650970/