Cell confluence is the percentage of a culture surface covered by adherent cells. Transfection is the laboratory process of artificially introducing foreign nucleic acids, such as DNA or RNA, into cells to modify their function or study gene expression. The success of this procedure hinges significantly on the cell’s physiological state, which is directly linked to its confluence level. The widely accepted optimal confluence for most adherent cell lines at the moment of transfection is typically between 70% and 90%.
The Ideal Confluence Range for Transfection Success
The 70% to 90% confluence range represents a biological “sweet spot” where cells are in the exponential growth phase, also known as the log phase. During this phase, cells are actively dividing and metabolically robust, which is a prerequisite for efficient transfection. An actively dividing cell possesses the necessary cellular machinery and high metabolic activity to effectively take up and process the foreign genetic material.
Cells in the log phase exhibit higher rates of endocytosis, a process where the cell membrane engulfs external substances, including the transfection complex (the mixture of nucleic acid and delivery reagent). This vigorous membrane activity is advantageous for the uptake of the genetic payload. The cell’s healthy state also ensures it can better tolerate the toxicity associated with many chemical transfection reagents, leading to better post-transfection survival.
This range provides high cell density to maximize the yield of transfected cells while avoiding the growth-inhibitory signals of overcrowding. For stable transfection, the goal is to integrate the foreign DNA into the host cell’s genome. Since cells in the log phase are cycling rapidly, this provides ample opportunity for the DNA to be incorporated before cell division occurs.
Maintaining the culture in this optimal state requires careful planning. Cells are often plated 18 to 24 hours before the scheduled transfection. This timing allows the cells to recover from the stress of passaging and re-adhere, ensuring they are in peak physiological condition. The goal is to catch the cells just as they are reaching maximum growth capacity but before they become too crowded.
Why Under-Confluence Hinders Efficiency
When cells are plated too sparsely, typically below 50% confluence, they enter a state of stress or quiescence that severely limits transfection efficiency. Cells are social entities that rely on communication with their neighbors to maintain proper function. This communication is facilitated by paracrine signaling, where cells secrete signaling molecules that influence the behavior of nearby cells.
In under-confluent cultures, the lack of sufficient cell-to-cell contact disrupts paracrine signaling, often referred to as a failure of quorum sensing. The absence of these growth-promoting signals causes cells to slow their metabolism, putting them into a temporary resting state. Cells in this quiescent state have significantly reduced endocytic activity, which dramatically lowers the efficiency of taking up the transfection complexes.
Sparsely plated cells are also highly susceptible to “edge effects,” where cells at the periphery experience different growth conditions than those in the center. The stress from sparse plating and low metabolic activity means the cells are less equipped to handle the toxicity of the transfection reagents. This combination of low uptake and poor viability results in a low number of successfully transfected cells and poor experimental yield.
Why Over-Confluence Reduces Viability
Transfecting cells at a confluence level exceeding 90% negatively impacts both efficiency and viability. The primary issue is the onset of contact inhibition, a natural regulatory mechanism that causes most normal adherent cells to cease proliferation upon physical contact. This contact triggers intracellular signals that halt the cell cycle.
A cell that has stopped actively dividing, or is entering the stationary phase, has a significantly reduced metabolic rate and low membrane activity. This metabolic slowdown reduces the cell’s ability to take up the transfection complex, resulting in low transfection efficiency. For stable transfection, the lack of mitotic activity prevents the introduced genetic material from being efficiently integrated into the host genome.
High cell density rapidly depletes nutrients and leads to the accumulation of toxic metabolic waste products. This creates a stressful, unfavorable environment for the cells, weakening their overall health. When the additional stress and toxicity of the transfection reagents are introduced, these compromised cells are highly likely to undergo cell death, or apoptosis. This results in poor post-transfection viability and a low yield of living, expressing cells.
Adjusting Confluence for Different Cell Lines and Methods
While the 70% to 90% range is a reliable starting point, the optimal confluence must be adjusted based on the specific cell type and transfection method used. Robust cell lines, such as HEK293 or HeLa, are generally tolerant of higher confluence, often performing well at the upper end of the optimal range. Conversely, more sensitive cells, like primary cells or stem cells, frequently require a slightly lower confluence (60% to 75%) to minimize stress and maximize viability.
The transfection technique itself also dictates the ideal cell state. Lipofection, a common chemical method relying on lipid-based reagents, typically performs best with adherent cells in the 70% to 90% range. This ensures sufficient cell surface area for the lipid-DNA complexes to interact. However, physical methods like electroporation often involve harvesting and concentrating the cells into a suspension immediately before the procedure.
Electroporation uses an electrical pulse to create transient pores in the cell membrane. This makes the confluence of the plated culture less relevant than the cell density in the suspension. The focus shifts to ensuring the cells are healthy and viable before they are harvested. Ultimate success is determined by empirical optimization, where researchers test a small range of confluence levels to find the specific conditions that yield the best balance of efficiency and cell survival.