Small volume bioreactors with substantially constant working volumes and associated systems and methods

A method of operating a bioreactor, comprising:

• performing for a period of time, within the bioreactor, a biochemical reaction in which at least one eukaryotic cell is grown within a liquid medium having a volume of less than about 50 milliliters, wherein the bioreactor comprises at least one online sensor that is in contact with the liquid medium for monitoring viability of the at least one eukaryotic cell, a gas inlet conduit and a gas outlet conduit connected to the bioreactor, and a gas bypass conduit that connects the gas inlet conduit and the gas outlet conduit, wherein the gas bypass conduit is external to the bioreactor, and wherein, during the period of time, a pressure gradient is applied to an inlet of the bioreactor using a pump and/or to an outlet of the bioreactor using a syringe;
• adding a first volume of liquid to the liquid medium in the bioreactor during a first period during the period of time;
• removing a second volume of liquid having a volume that is within 10% of the first volume of liquid from the liquid medium in the bioreactor during a second period during the period of time, wherein the first period and the second period do no overlap;
• repeating the adding and removing steps at least one time during the period of time;
• and
• monitoring the volume of liquid within the bioreactor during period of time;
• wherein:
• the adding step and the removing step are performed such that, between the adding step and the removing step, substantially no liquid is removed from the bioreactor via a nonevaporative pathway, and substantially no liquid is added to the bioreactor during the period of time;
• and
• over the entire period of time, the osmolarity of liquid within the bioreactor is maintained within a range of from about 200 osmoles per kilogram of the liquid to about 600 osmoles per kilogram of the liquid.