Run a UV sterilization cycle before starting hydrogel preparation. This step helps prepare the chamber environment before automated dispensing and mixing.

Record the initial chamber condition before formulation begins. This may include ambient chamber environment information available from the NSL system. This record can help compare hydrogel batches prepared under different laboratory conditions.

Turn on chamber illumination to support camera-based observation of the formulation vessel during reagent addition and hydrogel formation.

Dispense deionized water into the formulation vessel to set the initial aqueous phase for hydrogel preparation.

Add the chitosan solution as the main biopolymer component of the hydrogel. The chitosan solution should be prepared before the automated run and loaded into the assigned reservoir channel.

Start moderate stirring to mix the chitosan solution with the aqueous phase. Avoid very high stirring speed because it may introduce air bubbles into the developing gel matrix.

Apply mild heating to support polymer hydration and early viscosity development. The temperature should remain mild so that the formulation does not dry, degrade, or form uneven gel zones.

Hold the chitosan solution for a defined hydration period. This step allows the polymer phase to become more uniform before additional formulation components are added.

Add a small quantity of humectant solution to improve softness, spreadability, moisture retention, and handling behavior of the hydrogel prototype.

Add an optional hydrogel-supporting polymer such as sodium alginate, HPMC, gelatin, or another suitable polymer solution. This step can be used to modify the gel texture, matrix structure, and formulation consistency.

Mix the chitosan phase, humectant, and optional secondary polymer under controlled conditions. Low-to-moderate stirring is preferred to maintain uniformity while reducing bubble formation.

Add a mild stabilizing or conditioning solution according to the selected hydrogel design. This step may support gel network development, p H adjustment, or formulation stability. Exact p H confirmation should be performed offline because automated p H monitoring is not available as an NSL module.

Continue gentle mixing after conditioning solution addition. This supports uniform gel formation while minimizing air entrapment and mechanical disruption of the developing hydrogel network.

Keep the formulation undisturbed for gel maturation. This step allows viscosity development, polymer network stabilization, and reduction of mixing-induced bubbles.

Use mild sonication only if required for dispersion improvement or reduction of loose non-uniformity. This step should be used carefully because excessive sonication may disturb gel structure.

If sonication is used, mild bath temperature control may be applied where suitable. This step is optional and should be avoided if the hydrogel is already stable and uniform.

Use chamber illumination and camera recording to document the final hydrogel appearance. Observe general clarity, gel uniformity, air bubbles, visible lumps, phase separation, and flow behavior. This is a visual documentation step, not a UV-Vis or quantitative optical measurement.

Use exhaust control if required during formulation handling, mild heating, or acidic solution handling. This step supports chamber airflow management during the run.

Remove the prepared hydrogel from the formulation vessel and transfer it into a clean labelled container. Record batch ID, formulation composition, preparation time, and visual observations.

Perform further testing outside the NSL workflow as needed. Suggested tests include p H measurement, swelling behavior, spreadability, viscosity/rheology, stability observation, drug-loading study, antimicrobial testing, cytotoxicity testing, or biocompatibility evaluation. Methodology The chitosan-based bio-hydrogel was prepared using a Protoly-managed workflow supported by selected NSL hardware modules. Before formulation, the chamber was exposed to a timed UV sterilization step. The initial chamber condition was recorded using the available environment sensor module, and white chamber illumination was activated to support visual monitoring during the run. Deionized water was dispensed into the formulation vessel using the reservoir dispensing module. Pre-prepared chitosan solution was then added as the primary biopolymer phase. The mixture was stirred at moderate speed to promote uniform distribution of the polymer in the aqueous phase. Mild heating was applied using the heater module to assist polymer hydration and early viscosity development. After the hydration period, a small amount of glycerol or humectant solution was dispensed to improve the handling properties of the hydrogel prototype. Where required, an optional secondary polymer solution such as sodium alginate, HPMC, gelatin, or another hydrogel-supporting polymer was added to modify the hydrogel structure and consistency. The formulation was mixed under controlled low-to-moderate stirring conditions to reduce non-uniformity while avoiding unnecessary bubble formation. A mild stabilizing or p H-conditioning solution was then added according to the selected formulation design. Since p H monitoring is not an automated NSL module, accurate p H confirmation was treated as an offline/manual verification step. After this addition, the hydrogel precursor was gently stirred and then kept undisturbed for a defined maturation period. This allowed the hydrogel network to develop further and helped improve the apparent consistency of the formulation. Where required, mild sonication was used as an optional support step for dispersion improvement. The final hydrogel was visually documented using LED illumination and the camera module. The prepared hydrogel was then manually removed, labelled, and stored for offline characterization. Further studies such as p H measurement, swelling behavior, rheology, stability testing, antimicrobial evaluation, cytotoxicity assessment, and biocompatibility testing were considered downstream external evaluations. This workflow is intended for research-scale hydrogel preparation, formulation screening, and educational demonstration. It should not be considered a validated clinical or therapeutic hydrogel manufacturing process.